Acoustic perfusion devices

ABSTRACT

Acoustic perfusion devices for separating biological cells from other material in a fluid medium are disclosed. The devices include an inlet port, an outlet port, and a collection port that are connected to an acoustic chamber. An ultrasonic transducer creates an acoustic standing wave in the acoustic chamber that permits a continuous flow of fluid to be recovered through the collection port while keeping the biological cells within the acoustic chamber to be returned to the bioreactor from which the fluid medium is being drawn.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 62/256,952, filed on Nov. 18, 2015, and to U.S. ProvisionalPatent Application Ser. No. 62/243,211, filed on Oct. 19, 2015, and toU.S. Provisional Patent Application Ser. No. 62/211,057, filed on Aug.28, 2015, and to U.S. Provisional Patent Application Ser. No.62/093,491, filed on Dec. 18, 2014. This application is also acontinuation-in-part of U.S. patent application Ser. No. 14/175,766,filed on Feb. 7, 2014, which claims priority to U.S. Provisional PatentApplication Ser. No. 62/761,717, filed on Feb. 3, 2013, and is also acontinuation-in-part of U.S. patent application Ser. No. 14/026,413,filed on Sep. 13, 2013, which claims the benefit of U.S. ProvisionalPatent Application Ser. No. 61/708,641, filed on Oct. 2, 2012. U.S.patent application Ser. No. 14/026,413 is also a continuation-in-part ofU.S. Ser. No. 13/844,754, filed Mar. 15, 2013, which claims the benefitof U.S. Provisional Patent Application Ser. No. 61/611,159, filed Mar.15, 2012, and of U.S. Provisional Patent Application Ser. No.61/611,440, filed Mar. 15, 2012, and of U.S. Provisional PatentApplication Ser. No. 61/754,792, filed Jan. 21, 2013. These applicationsare incorporated herein by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No.IIP-1330287 (Amendment 003, Proposal No. 1458190) awarded by theNational Science Foundation. The government has certain rights in theinvention.

BACKGROUND

The field of biotechnology has grown tremendously in the last 20 years.This growth has been due to many factors, some of which include theimprovements in the equipment available for bioreactors, the increasedunderstanding of biological systems and increased knowledge as to theinteractions of materials (such as monoclonal antibodies and recombinantproteins) with the various systems of the human body.

Improvements in equipment have allowed for larger volumes and lower costfor the production of biologically derived materials such as recombinantproteins. This is especially prevalent in the area of pharmaceuticals,where the successes of many types of new drug therapies have beendirectly due to the ability to mass produce these materials throughprotein-based manufacturing methods.

One of the key components that is utilized in the manufacturingprocesses of new biologically based pharmaceuticals is the bioreactorand the ancillary processes associated therewith. An area of growth inthe bioreactor field has been with the perfusion process. The perfusionprocess is distinguished from the fed-batch process by its lower capitalcost and continuous (rather than batch) operation.

In the fed-batch process, a culture is seeded in a bioreactor. Thegradual addition of a fresh volume of selected nutrients during thegrowth cycle is used to improve productivity and growth. The product isrecovered after the culture is harvested. The discontinuous fed-batchbioreactor process has been attractive because of its simplicity andalso due to carryover from well-known fermentation processes. However, afed-batch bioreactor has high start-up costs, and generally has a largevolume to obtain a cost-effective amount of product at the end of thegrowth cycle. After the batch is completed, the bioreactor must becleaned and sterilized, resulting in nonproductive downtime.

A perfusion bioreactor processes a continuous supply of fresh media thatis fed into the bioreactor while growth-inhibiting byproducts areconstantly removed. The nonproductive downtime can be reduced oreliminated with a perfusion bioreactor process. The cell densitiesachieved in perfusion culture (30-100 million cells/mL) are typicallyhigher than for fed-batch modes (5-25 million cells/mL). Theseimprovements have led to lower contamination in the harvest and betteryields without significant increase in cost. However, a perfusionbioreactor requires a cell retention device to prevent escape of theculture when byproducts are being removed. These cell retention systemsadd a level of complexity to the perfusion process, requiringmanagement, control, and maintenance for successful operation.Operational issues such as malfunction or failure of the cell retentionequipment has previously been a problem with perfusion bioreactors,which has limited their attractiveness in the past.

BRIEF DESCRIPTION

The present disclosure relates, in various embodiments, to acousticdevices which are used for perfusion biomanufacturing. Moreparticularly, the devices are coupled to an associated bioreactor.Within the bioreactor, biomolecules, such as recombinant proteins ormonoclonal antibodies, are produced. The acoustic device is then usedfor separating these desirable products from the cells on a continuousbasis, and the cells are continuously returned to the bioreactor.Generally, a fluid medium containing the cells and the desired productsare passed or flowed through the acoustic device and separated thereinby multi-dimensional standing wave(s). The fluid medium can becontinuously flowed into the device, with desired products beingcontinuously removed. The acoustic perfusion device returns healthyviable cells to the bioreactor while desired products are harvested andflowed downstream for further processing, e.g., additional filtering,chromatography, etc. Additionally, the cell culture media in thebioreactor is clarified as cell fragments are also allowed to pass intothe harvest stream and thereby out of the fluid medium being recycled tothe bioreactor. This results in lower overall cell culture media usage,corresponding to a predicted cost savings of up to $20,000 per day forlarge bioreactors.

Disclosed in various embodiments are acoustic perfusion devices,comprising: an acoustic chamber; an inlet port, an inlet flow pathleading from the inlet port to the acoustic chamber; an outlet port forrecirculating fluid flowing through the device back to its source (e.g.a bioreactor); at least one collection or harvest port for collecting aproduct stream of fluid exiting the acoustic chamber; and at least oneultrasonic transducer in the acoustic chamber below the at least oneharvest port, the at least one ultrasonic transducer including apiezoelectric material driven by a voltage signal to create an acousticstanding wave across a collection or harvest flow path leading from theacoustic chamber to the at least one collection or harvest port. Theacoustic standing wave may be planar or multi-dimensional, or acombination of such waves may be present within the acoustic chamber(generally from multiple transducers). The acoustic standing wave can bethought of as a “force field” that holds back whole cells but permitssmaller materials such as the desired biomolecules (e.g. recombinantproteins and/or monoclonal antibodies) and cell fragments, to passthrough and be removed from the fluid that is returned to thebioreactor.

The outlet port is generally below the inlet port, and is generallylocated at a bottom end of the device.

As mentioned above, the device may have one or more collection orharvest ports at the top of the device. In some more specificembodiments, the device may have a total of two harvest ports spacedapart from each other on the top end of the device.

In particular embodiments, the inlet port is at a first end of thedevice at a first height, the at least one ultrasonic transducer is at asecond height above the first height, and a bottom wall extends from theinlet port to the outlet port. The outlet port may be located at asecond end of the device opposite the first end. The bottom wall may beconcave, relative to a line between the inlet port and the outlet port.The device may include an upper wall above the inlet flow path. Theinlet port, the outlet port, and the at least one harvest port aresometimes all located on a front wall of the device. The front wallitself may be planar (i.e. flat).

The device can further comprise a reflector located in the acousticchamber opposite the at least one ultrasonic transducer. Alternatively,the device can have a total of two ultrasonic transducers located onopposite sides of the harvest flow path at the same height and facingeach other, or additional ultrasonic transducers can be located onmultiple sides of the collection/harvest flow path. A reflector may belocated between the two ultrasonic transducers. There may also be aplurality of transducer/reflector pairs located as appropriate to formplanar, multi-dimensional, or combinations of such acoustic standingwave(s).

In particular embodiments, the acoustic standing wave results in anacoustic radiation force having an axial force component and a lateralforce component that are of the same order of magnitude.

In other embodiments of the device disclosed herein, the inlet flow pathleads from the inlet port downwards towards a bottom end of the deviceand past the outlet port, and then upwards to the acoustic chamber.Sometimes, the inlet port and the at least one harvest port are bothlocated on a top wall of the device, and the outlet port is located on afront wall of the device. The at least one ultrasonic transducer may bemounted in a rear wall or a front wall of the device. The bottom wall ofthis acoustic chamber can be a sloped planar surface. The reflector maybe made of a transparent material.

The inlet flow path may be shaped to generate a tangential flow pathbelow an acoustic field generated by the acoustic standing wave. Instill additional versions seen herein, the inlet flow path enters theacoustic chamber on a first side of the device, and the outlet port islocated (i) on the first side of the device or (ii) on a second oppositeside. The inlet port can be located on a front side of the device, andthe at least one harvest port can be located on a top wall of thedevice. The at least one transducer can be located on a front side or arear side of the device. In more particular embodiments, there can betwo transducers, one on the front side and one of the rear side. In yetother particular embodiments, there is an ultrasonic transducer on thefront or rear side, and a reflector located on the respective rear orfront side opposite the transducer.

The device may be attached to a mounting piece having holes forattachment.

Also disclosed are methods for separating cells from a fluid mediumcontaining the cells. The fluid medium is flowed through an acousticperfusion device of the structure described above, having at least oneultrasonic transducer. The at least one ultrasonic transducer is drivento create the acoustic standing wave. A fluid enriched in cells can becollected from the outlet port and a clarified fluid, depleted in cells,can be collected from the at least one harvest port.

In particular embodiments, the flow rate through the collection/harvestflow path is at least one order of magnitude smaller than a flow ratethrough the inlet flow path. In specific embodiments, a flow rate of thefluid medium entering the device through the inlet port is about 1 literper minute and a flow rate of the fluid depleted in cells exiting thedevice through the at least one collection/harvest port is about 10milliliters per minute. Alternatively, the ratio of the flow rateentering through the inlet port to the flow rate exiting through the atleast one collection/harvest port is such that the acoustic standingwave is not overcome by the main body of cells, or in other words sothat a large volume of cells do not begin exiting the device through thecollection/harvest port(s).

The methods may further comprise pulling the fluid medium through thedevice using a first pump attached to the at least one harvest port ofthe device and a second pump attached to the outlet port of the device.

Also disclosed herein are flow devices adapted to (i) receive a flowingmixture containing a primary fluid and cells; and (ii) to use a firstacoustic standing wave to continuously draw off a harvest fluid streamdepleted in cells from the flowing mixture, thereby changing the cellconcentration of the flowing mixture. A pressure rise may be generatedat an edge of the acoustic standing wave, along with an acousticradiation force. This “edge effect” acts as a barrier at the edge of theacoustic standing wave. The frequency of the acoustic standing wave maybe modified such that different contrast factor materials may be heldback or allowed through the acoustic standing wave.

The device may further comprise a secondary flow chamber in which theharvest fluid stream depleted in cells passes through a second acousticstanding wave having a frequency higher than the first acoustic standingwave.

Also disclosed herein are flow devices that comprise: at least one inletfor receiving a flowing mixture of a primary fluid and cells, anultrasonic transducer that produces a first ultrasonic acoustic standingwave and uses a pressure rise and an acoustic radiation force generatedat an edge of the first ultrasonic acoustic standing wave to separatethe flowing mixture into a primary high cell concentration fluid streamand a secondary harvest fluid stream; an outlet port for the primaryhigh cell concentration fluid stream; and at least one collection portfor the secondary harvest fluid stream. A bleed port can also be presentfor extracting a concentrated fluid/cell mixture.

The device may further comprise a secondary flow chamber in which thesecondary harvest fluid stream passes through a second acoustic standingwave having a frequency higher than the first ultrasonic acousticstanding wave.

These and other non-limiting characteristics are more particularlydescribed below.

BRIEF DESCRIPTION OF THE DRAWINGS

The following is a brief description of the drawings, which arepresented for the purposes of illustrating the exemplary embodimentsdisclosed herein and not for the purposes of limiting the same.

FIG. 1 illustrates a single standing acoustic wave generated by anultrasonic transducer and a reflector.

FIG. 2 is an illustration comparing a fed-batch bioreactor system with aperfusion bioreactor system.

FIG. 3 is a cross-sectional view that shows the various components of astirred-tank bioreactor.

FIG. 4 is a perspective view of one exemplary embodiment of an acousticperfusion device of the present disclosure, having two collection orharvest ports and a single ultrasonic transducer.

FIG. 5 shows a second exemplary embodiment of an acoustic perfusiondevice of the present disclosure, with a single reflector locatedbetween two ultrasonic transducers.

FIG. 6 is a schematic view illustrating a perfusion bioreactor coupledwith an acoustic perfusion device of the present disclosure, and arecycle path.

FIG. 7 is a front cross-sectional view of a third exemplary embodimentof an acoustic perfusion device of the present disclosure.

FIG. 8 is an exterior perspective view of the acoustic perfusion deviceof FIG. 7.

FIG. 9 is a front cross-sectional view of a fourth exemplary embodimentof an acoustic perfusion device of the present disclosure.

FIG. 10 is a perspective view of the acoustic perfusion device of FIG.9.

FIG. 11 is a cross-sectional diagram of a conventional ultrasonictransducer.

FIG. 12 is a cross-sectional diagram of an ultrasonic transducer of thepresent disclosure. An air gap is present within the transducer, and nobacking layer or wear plate are present.

FIG. 13 is a cross-sectional diagram of an ultrasonic transducer of thepresent disclosure. An air gap is present within the transducer, and abacking layer and wear plate are present.

FIG. 14 is a graph of electrical impedance amplitude versus frequencyfor a square transducer driven at different frequencies.

FIG. 15 illustrates the trapping line configurations for seven of theresonance frequencies (minima of electrical impedance amplitudes) ofFIG. 14 from the direction orthogonal to fluid flow.

FIG. 16 is a computer simulation of the acoustic pressure amplitude(right-hand scale in Pa) and transducer out of plane displacement(left-hand scale in meters). The text at the top of the left-hand scalereads “×10⁻⁷”. The text at the top of the left-hand scale by theupward-pointing triangle reads “1.473×10⁻⁶”. The text at the bottom ofthe left-hand scale by the downward-pointing triangle reads“1.4612×10⁻¹⁰”. The text at the top of the right-hand scale reads“×10⁶”. The text at the top of the right-hand scale by theupward-pointing triangle reads “1.1129×10⁶”. The text at the bottom ofthe right-hand scale by the downward-pointing triangle reads “7.357”.The triangles show the maximum and minimum values depicted in thisfigure for the given scale. The horizontal axis is the location withinthe chamber along the X-axis, in inches, and the vertical axis is thelocation within the chamber along the Y-axis, in inches.

FIG. 17 shows the In-Plane and Out-of-Plane displacement of a crystalwhere composite waves are present.

FIG. 18 is a view of a first acoustic perfusion device of the presentdisclosure fluidly connected to an associated bioreactor, showing aplurality of hoses fluidly connecting the various ports of the device tothe associated bioreactor and an outflow pump fluidly connecting theoutlet port of the device to the associated bioreactor.

FIG. 19 is a view of another acoustic perfusion device of FIG. 5,showing a reflector in the acoustic chamber between first and secondtransducers. A fluid medium is also present in the device and arrows areshown indicating the direction of flow in addition to waves indicatingthe acoustic field between the reflector and first and secondtransducers.

FIG. 20 is a graph showing the efficiency of removing cells from a fluidmedium for one experiment at two different perfusate/feed rates.

FIG. 21 is a graph showing the harvest flow (also referred to as theperfusate) turbidity reduction for an experiment

FIG. 22 is a graph showing the cell viability for varied flow rates forthe experiment conducted for the graphs of FIGS. 20-21.

FIG. 23 is a graph showing the total cell density and cell retention forvaried flow rates and flow methods for another experiment.

FIG. 24 is a graph showing the cell viability for varied flow rates forthe experiment conducted for the graphs of FIG. 23.

FIG. 25 is a graph showing the total cell density and cell retention forvaried numbers of ultrasonic transducers for another experiment.

FIG. 26 is a graph showing the cell viability for varied numbers ofultrasonic transducers for the experiment conducted for the graph ofFIG. 25.

FIG. 27 is a picture of another acoustic perfusion device that wastested.

FIG. 28 is a graph showing the effect of the perfused flow rate or thetransducer voltage on the cell retention.

FIG. 29A is a microscope image from a ViCell Cell Analyzer of theparticles in the feed stream going into the device. The feed stream is abioreactor fluid containing CHO cells, protein, and cell fragments. FIG.29B is a graph of the particle diameter distribution of the feed,showing a bimodal distribution. In FIG. 29B, the y-axis is the particlecount from 0 to 200 in intervals of 10. The x-axis is the particlediameter in microns from 6 to 50 in intervals of 2. The total number ofparticles is 5539, the mean particle size is 16.78 microns, standarddeviation of 6.76 microns, and mode particle size of 10.56 microns.

FIG. 30A is a microscope image of the perfusate (or clarified harvestflow) exiting the device. FIG. 30B is a graph of the particle diameterdistribution of the perfusate, showing a unimodal distribution at muchlower sizes. In FIG. 30B, the y-axis is the particle count from 0 to 300in intervals of 20. The x-axis is the particle diameter in microns from6 to 50 in intervals of 2. The total number of particles is 2919, themean particle size is 10.08 microns, standard deviation of 3.75 microns,and mode particle size of 8.99 microns.

FIG. 31 is a CFD model showing the velocity distribution within thedevice of FIG. 27. The text at the top of the scale reads “×10⁻²”. Thetext at the top of the scale by the upward-pointing triangle reads“0.678”. The text at the bottom of the scale by the downward-pointingtriangle reads “0”. The triangles show the maximum and minimum valuesdepicted in this figure for the given scale. The scale runs from 0 to 5m/s in intervals of 0.5, with black indicating 5 at the top of thescale, and white indicating zero at the bottom of the scale.

FIG. 32 is a front view of the device of FIG. 27, showing the flowpaths, acoustic field, and acoustic edge effect.

FIG. 33 is a composite photograph showing the acoustic perfusion deviceof FIG. 27 in two operating modes. On the left, the device is in startupor cell settling mode. On the right, the device is in steady cellretention mode.

FIG. 34 shows the geometry of a model simulation of the acoustic deviceused for cell retention. The model contains two fluids, one a clarifiedfluid within the acoustic field, the other a high cell density fluid tothe left of the acoustic field, a piezoelectric transducer, a steelreflector, and an aluminum housing. The first fluid was water within theacoustic field and the second fluid was a 15% concentration of CHO cellsin water solution outside (to the left) of the acoustic field. The bluesolid line in the model indicates the separation line between the twofluids.

FIGS. 35A, 35B, and 35C are graphs showing the displacement of thepiezoelectric material, the aluminum housing, and the steel reflector(left-side scale); and the acoustic pressure in the two fluids(right-side scale) of the model simulation of FIG. 34 at severalfrequencies of operation. FIG. 35A is at a frequency of 2.218 MHz. FIG.35B is at a frequency of 2.2465 MHz. FIG. 35C is at a frequency of2.3055 MHz. For all three graphs, the left-side scale is indicated withtext at the top of the scale reading “×10⁻⁶” or “×10⁻⁷”, and is in unitsof inches. The right-side scale is indicated with text at the top of thescale reading “×10⁶”, and is in units of Pascals. The y-axis runs from−0.8 to 1.6 in intervals of 0.2. The x-axis runs from −0.5 to 1.5 inintervals of 0.5.

FIG. 36 is a graph showing the average lateral force (N) and the averagelateral force normalized by power (N/W) acting on suspended CHO cells atseveral frequencies of operation.

FIG. 37 is a picture (top view) of an acoustic perfusion device of thepresent disclosure. Arrows indicate the flow into the inlet port; theflow out of the outlet port; the clarified fluid flow out the top of thedevice and the flow of concentrate out the bottom of the device.

FIG. 38 is a picture (side view) of the acoustic perfusion device ofFIG. 37.

FIG. 39 is a graph of cell retention vs. perfusate flowrate for thedevice of FIG. 37.

FIG. 40 is a prior art illustration showing direct flow filtration (DFF)and tangential flow filtration (TFF).

FIG. 41 is a picture illustrating a first mode of operation duringperfusion, in which cells are trapped, clustered, and separated from aharvest stream. The device is operated vertically, with an arrowindicating the direction of gravity.

FIG. 42 is a picture illustrating a second mode of operation duringperfusion, in which cells are prevented from entering an acousticstanding wave field while smaller particles are permitted to passthrough the field and into the harvest stream. The device is operatedvertically, with an arrow indicating the direction of gravity.

DETAILED DESCRIPTION

The present disclosure may be understood more readily by reference tothe following detailed description of desired embodiments and theexamples included therein. In the following specification and the claimswhich follow, reference will be made to a number of terms which shall bedefined to have the following meanings.

Although specific terms are used in the following description for thesake of clarity, these terms are intended to refer only to theparticular structure of the embodiments selected for illustration in thedrawings, and are not intended to define or limit the scope of thedisclosure. In the drawings and the following description below, it isto be understood that like numeric designations refer to components oflike function.

The singular forms “a,” “an,” and “the” include plural referents unlessthe context clearly dictates otherwise.

The term “comprising” is used herein as requiring the presence of thenamed component and allowing the presence of other components. The term“comprising” should be construed to include the term “consisting of”,which allows the presence of only the named component, along with anyimpurities that might result from the manufacture of the namedcomponent.

Numerical values should be understood to include numerical values whichare the same when reduced to the same number of significant figures andnumerical values which differ from the stated value by less than theexperimental error of conventional measurement technique of the typedescribed in the present application to determine the value.

All ranges disclosed herein are inclusive of the recited endpoint andindependently combinable (for example, the range of “from 2 grams to 10grams” is inclusive of the endpoints, 2 grams and 10 grams, and all theintermediate values). The endpoints of the ranges and any valuesdisclosed herein are not limited to the precise range or value; they aresufficiently imprecise to include values approximating these rangesand/or values.

The modifier “about” used in connection with a quantity is inclusive ofthe stated value and has the meaning dictated by the context. When usedin the context of a range, the modifier “about” should also beconsidered as disclosing the range defined by the absolute values of thetwo endpoints. For example, the range of from about 2 to about 10″ alsodiscloses the range “from 2 to 10.” The term “about” may refer to plusor minus 10% of the indicated number. For example, “about 10%” mayindicate a range of 9% to 11%, and “about 1” may mean from 0.9-1.1.

It should be noted that many of the terms used herein are relativeterms. For example, the terms “upper” and “lower” are relative to eachother in location, i.e. an upper component is located at a higherelevation than a lower component in a given orientation, but these termscan change if the device is flipped. The terms “inlet” and “outlet” arerelative to a fluid flowing through them with respect to a givenstructure, e.g. a fluid flows through the inlet into the structure andflows through the outlet out of the structure. The terms “upstream” and“downstream” are relative to the direction in which a fluid flowsthrough various components, i.e. the flow fluids through an upstreamcomponent prior to flowing through the downstream component. It shouldbe noted that in a loop, a first component can be described as beingboth upstream of and downstream of a second component.

The terms “horizontal” and “vertical” are used to indicate directionrelative to an absolute reference, i.e. ground level. However, theseterms should not be construed to require structures to be absolutelyparallel or absolutely perpendicular to each other. For example, a firstvertical structure and a second vertical structure are not necessarilyparallel to each other. The terms “top” and “bottom” or “base” are usedto refer to surfaces where the top is always higher than the bottom/baserelative to an absolute reference, i.e. the surface of the earth. Theterms “upwards” and “downwards” are also relative to an absolutereference; upwards is always against the gravity of the earth.

The present application refers to “the same order of magnitude.” Twonumbers are of the same order of magnitude if the quotient of the largernumber divided by the smaller number is a value less than 10.

Bioreactors are useful for making biomolecules such as recombinantproteins or monoclonal antibodies. Very generally, cells are cultured ina bioreactor vessel with media in order to produce the desired product,and the desired product is then harvested by separation from the cellsand media in an acoustic perfusion device, such as the device of thepresent disclosure. The acoustic filtering device permits the withdrawalof some desired product, a small portion of the media, and cellularfragments/debris smaller than the cells, with the remainder beingrecycled back to the bioreactor (particularly the cells). The use ofmammalian cell cultures including Chinese hamster ovary (CHO), NSOhybridoma cells, baby hamster kidney (BHK) cells, insect cells, andhuman cells (e.g. T-cells, B-cells, stem cells, red blood cells), andliving/biological cells in general has proven to be a very efficaciousway of producing/expressing the recombinant proteins and monoclonalantibodies used in various applications such as pharmaceuticals orvaccines. Two general types of bioreactor processes exist: fed-batch andperfusion.

While fed-batch reactors are the norm currently, due mainly to thefamiliarity of the process to many scientists and technicians, perfusiontechnology is growing at a very fast rate. Many factors favor the use ofa perfusion bioreactor process, primarily because it is conducive tocontinuous production. The capital and start-up costs for perfusionbioreactors are lower, smaller upstream and downstream capacity isrequired, throughput can be higher, the process is continuous, and theprocess uses smaller volumes and fewer seed steps than fed-batchmethods. A perfusion bioreactor process also lends itself better todevelopment, scale-up, optimization, parameter sensitivity studies, andvalidation.

A perfusion bioreactor may also be utilized to generate cells that wouldbe utilized in a cell therapy process. In this type of perfusionbioreactor, biological cells such as CAR T-cells, Jurkat T-cells and thelike are cultured in a perfusion bioreactor. The acoustic standing waveused in the perfusion devices of the present disclosure can be used toseparate viable and nonviable cells after the transfection process. Thisallows for improved efficacy of the inoculation of the patient with thisT-cell therapy as only viable cells are utilized. The nonviable cellsand cell fragments are separated out through the perfusion process, withthese materials going into the secondary flow and exiting thebioreactor.

Recent developments in perfusion bioreactor technology also favor itsuse. Control technology and general support equipment is improving forperfusion bioreactors, increasing the robustness of perfusion processes.The perfusion process can now be scaled up to bioreactors having avolume up to 1000 liters (L). Better cell retention systems forperfusion bioreactors result in lower cell loss and greater celldensities than have been seen previously. Cell densities greater than 50million cells/mL are now achievable, compared to fed-batch celldensities of around 20 million cells/mL. Lower contamination andinfection rates have improved the output of perfusion bioreactors.Higher product concentrations in the harvest and better yields withoutsignificant increase in cost have thus resulted for perfusion processes.

Perfusion bioreactors are particularly attractive because of thecontinuous production of the biomolecules from the expressing cellculture, and shorter residence time of said biomolecules in the processprior to harvest. The target cells are held back by a filtrationprocess, such as tangential flow filtration (TFF) or alternatingtangential flow filtration (ATF) while the expressed biomolecules areextracted from the perfusion bioreactor. The cells are then returned tothe bioreactor to ensure they receive the nutrition and oxygen tomaintain the production of the overall cell culture. In the perfusionreactor process, the cells continue to multiply so it is also necessaryto bleed off some of the cell culture population throughout theperfusion production process.

The TFF and ATF processes of filtration have several issues, such asclogging/fouling and loss of biomolecule product (particularly at highcell densities), all directly related to the nature of the hollow fibermembranes used in the filtration. It is therefore desirable to find anew filtration process that does not clog and minimizes loss of thedesired biomolecule product. In addition, TFF and ATF will retain allcellular debris and fines within the bioreactor, which is not desirable.A process capable of distinguishing between cell retention whileallowing for the passing of cell debris and fines may therefore befavorable.

Briefly, the present disclosure relates to acoustic perfusion devicescapable of generating multi-dimensional acoustic standing wave(s) fromone or more piezoelectric transducers, where the transducers areelectrically such that they move in a multi-mode displacement patternrather than a “piston” mode of vibration. Through this manner ofacoustic standing wave generation, a higher lateral trapping force isgenerated than if the piezoelectric transducer is excited in a “piston”mode where only one large standing wave is generated. Thus, with thesame input power to a piezoelectric transducer, the multi-dimensionalacoustic standing waves can have a higher lateral trapping forcecompared to a planar acoustic standing wave. The input power is tunablefor a controlled flow. This can be used to facilitate proteinaceousfluid purification of a fluid stream coming from a bioreactor.Alternatively, the acoustic standing wave may also be a planar standingwave where the piezoelectric transducer is excited in the piston mode,generating a planar wave. The acoustic standing wave(s) may also be acombination of planar and multi-dimensional acoustic standing waves. Allof these standing waves generate an “edge effect” such that the cellsfrom the bioreactor are held back and the biomolecule product expressedfrom the cells, cell fragments and small debris are allowed to passthrough.

Acoustophoresis is a low-power, no-pressure-drop, no-clog, solid-stateapproach to particle separation from fluid dispersions (i.e., it is usedto achieve separations that are more typically performed with porousfilters, but it has none of the disadvantages of filters). Inparticular, the acoustic perfusion devices of the present disclosure aresuitable for use with macro-scale bioreactors for separations in flowingsystems with high flow rates. The acoustic perfusion device is designedto create a high intensity multi-dimensional ultrasonic standing wavethat results in an acoustic radiation force that can overcome thecombined effects of fluid drag and buoyancy or gravity at low flowrates. As a result, the radiation force acts as a filter that preventstargeted particles (e.g., biological cells) from crossing the plane ofthe standing wave. As explained above, the trapping capability of astanding wave may be varied as desired, for example by varying the flowrate of the fluid, the acoustic radiation force, and the shape of theacoustic filtering device to maximize cell retention through trappingand settling. This technology offers a green and sustainable alternativefor separation of secondary phases with a significant reduction in costof energy. Excellent particle separation efficiencies have beendemonstrated for particle sizes as small as one micron.

Generally, the scattering of the acoustic field off the particlesresults in a three-dimensional acoustic radiation force, which acts as athree-dimensional trapping field. The acoustic radiation force isproportional to the particle volume (e.g., the cube of the radius) whenthe particle is small relative to the wavelength. It is proportional tofrequency and the acoustic contrast factor. It also scales with acousticenergy (e.g., the square of the acoustic pressure amplitude). Forharmonic excitation, the sinusoidal spatial variation of the force iswhat drives the particles to the stable positions within the standingwaves. When the acoustic radiation force exerted on the particles isstronger than the combined effect of fluid drag force andbuoyancy/gravitational force, the particle is trapped within theacoustic standing wave field. The action of the lateral and axialacoustic forces on the trapped particles results in formation of tightlypacked clusters through concentration, agglomeration and/or coalescenceof particles that settle through enhanced gravity (particles heavierthan the host fluid) or buoyancy (particles lighter than the hostfluid). Additionally, secondary inter-particle forces, such as Bjerknessforces, aid in particle agglomeration.

Most biological cell types present a higher density and lowercompressibility than the medium in which they are suspended, so that theacoustic contrast factor between the cells and the medium has a positivevalue. As a result, the axial acoustic radiation force (ARF) drives thecells towards the standing wave pressure nodes. The axial component ofthe acoustic radiation force drives the cells, with a positive contrastfactor, to the pressure nodal planes, whereas cells or other particleswith a negative contrast factor are driven to the pressure anti-nodalplanes. The radial or lateral component of the acoustic radiation forceis the force that traps the cells. The radial or lateral component ofthe ARF is larger than the combined effect of fluid drag force andgravitational force. For small cells or emulsions the drag force F_(D)can be expressed as:

${{\overset{\rightarrow}{F}}_{D} = {4\pi \; \mu_{f}{{R_{p}\left( {{\overset{\rightarrow}{U}}_{f} - {\overset{\rightarrow}{U}}_{p}} \right)}\left\lbrack \frac{1 + {\frac{3}{2}\hat{\mu}}}{1 + \hat{\mu}} \right\rbrack}}},$

where U_(f) and U_(p) are the fluid and cell velocity, R_(p) is theparticle radius, μ_(f) and μ_(p) are the dynamic viscosity of the fluidand the cells, and {circumflex over (μ)}={circumflex over (μ)}_(p)/μ_(f)is the ratio of dynamic viscosities. The buoyancy force F_(B) isexpressed as:

$F_{B} = {\frac{4}{3}\pi \; {R_{p}^{3}\left( {\rho_{f} - \rho_{p}} \right)}{g.}}$

For a cell to be trapped in the multi-dimensional ultrasonic standingwave, the force balance on the cell must be zero, and, therefore, anexpression for lateral acoustic radiation force F_(LRF) can be found,which is given by:

F _(LRF) =F _(D) +F _(B).

For a cell of known size and material property, and for a given flowrate, this equation can be used to estimate the magnitude of the lateralacoustic radiation force.

The theoretical model that is used to calculate the acoustic radiationforce is based on the formulation developed by Gor'kov. The primaryacoustic radiation force F_(A) is defined as a function of a fieldpotential U, F_(A)=−∇(U),

where the field potential U is defined as

${U = {V_{0}\left\lbrack {{\frac{\langle p^{2}\rangle}{2\rho_{f}c_{f}^{2}}f_{1}} - {\frac{3\rho_{f}{\langle u^{2}\rangle}}{4}f_{2}}} \right\rbrack}},$

and f₁ and f₂ are the monopole and dipole contributions defined by

${f_{1} = {1 - \frac{1}{{\Lambda\sigma}^{2}}}},{f_{2} = \frac{2\left( {\Lambda - 1} \right)}{{2\Lambda} + 1}},$

where p is the acoustic pressure, u is the fluid particle velocity, Λ isthe ratio of cell density ρ_(p) to fluid density ρ_(f), σ is the ratioof cell sound speed c_(p) to fluid sound speed c_(f), V_(o) is thevolume of the cell, and < > indicates time averaging over the period ofthe wave.

Gor'kov's theory is limited to particle sizes that are small withrespect to the wavelength of the sound fields in the fluid and theparticle, and it also does not take into account the effect of viscosityof the fluid and the particle on the radiation force. Additionalnumerical models have been developed for the calculation of the acousticradiation force for a particle without any restriction as to particlesize relative to wavelength. These models also include the effect offluid and particle viscosity, and therefore are a more accuratecalculation of the acoustic radiation force. The models that wereimplemented are based on the theoretical work of Yurii Ilinskii andEvgenia Zabolotskaya as described in AIP Conference Proceedings, Vol.1474-1, pp. 255-258 (2012).

Desirably, the ultrasonic transducer(s) generates a multi-dimensionalstanding wave in the fluid that exerts a lateral force on the suspendedparticles to accompany the axial force. Typical results published inliterature state that the lateral force is two orders of magnitudesmaller than the axial force. In contrast, the technology disclosed inthis application provides for a lateral force to be of the same order ofmagnitude as the axial force. However, in certain embodiments describedfurther herein, the device use both transducers that producemulti-dimensional acoustic standing waves and transducers that produceplanar acoustic standing waves. For purposes of this disclosure, astanding wave where the lateral force is not the same order of magnitudeas the axial force is considered a “planar acoustic standing wave.” Thelateral force component of the total acoustic radiation force (ARF)generated by the ultrasonic transducer(s) of the present disclosure issignificant and is sufficient to overcome the fluid drag force at linearvelocities of up to 1 cm/s, and to create tightly packed clusters, andis of the same order of magnitude as the axial force component of thetotal acoustic radiation force.

It may be helpful to contrast the technology of the present disclosurewith that of prior filtration technology. FIG. 40 shows two prior artfiltration methods. The left-hand side of FIG. 40 illustrates directflow filtration (DFF). In DFF, the entire feed stream 4005 of fluid andparticles is directed towards the filter. The filter 4010 holds back theparticles 4020 that are larger than the filter's pore size, whereassmaller particles 4030 and the fluid pass through the filter. Theright-hand side of FIG. 40 illustrates tangential flow filtration (TFF).In TFF, the feed stream is not directed towards the filter. Rather, thefeed stream is directed tangentially to the filter, such that a majorityof the feed stream passes tangentially over the filter surface.Typically, this feed stream is recirculated to pass by the filter morethan once. A much smaller filtrate stream 4006 is pulled through thefilter membrane containing the smaller particles 4030. One advantage ofTFF over DFF is that the tangential stream reduces the clogging andfouling of the filter and the formation of a gel layer that sits on topof the filter.

In the devices of the present disclosure, during startup, the fluidensonified by the acoustic standing wave is clarified by the process oftrapping cells and growing them into tightly packed clusters, such thatcontinuous gravitational separation of the clusters of cells takesplace. Since there is a limited amount of new cells flowing into thisvolume, this results in a rapid clarification of the fluid subjected tothe acoustic standing wave. When this state is reached, the system canbe described as including two fluids: a first fluid containing thedesired product and small cell fragments/debris (which has passedthrough the acoustic standing wave), and a second fluid containing thebioreactor fluid and all of the cells (which are held back by theacoustic standing wave). The two fluids may be of different effectiveacoustic properties, such as density and speed of sound, with awell-defined interface between these two fluids. The interface isgenerally located near the lower edge of the acoustic standing wavefield, generating an “acoustic edge effect”. The first fluid (i.e., thefluid that has been clarified and contains the product, some cells, andcell fragments) is downstream of the interface and represents theharvest flow and occupies the volume of fluid ensonified by the acousticstanding wave field. The second fluid (i.e., the fluid containing thebioreactor fluid and most of the cells) is upstream of the interface.These two different fluids can be seen in FIG. 33.

The acoustic standing wave field exerts an acoustic radiation pressure(i.e. a pressure rise) and a force at the acoustic edge of the acousticfield on the interface between the two fluids, thereby keeping theupstream cells from entering the acoustic field. The occurrence of theradiation pressure and the force on the interface allows for the firstfluid containing the product to pass through the interface whileretaining the cells in the upstream fluid. The cells that are held backby the effect of the acoustic radiation force at the interface betweenthe two fluids can be continuously returned to the bioreactor to ensurethey receive the nutrition and oxygen to maintain the production of theoverall cell culture.

The circulating motion of the flow field underneath the interfacetransports the cells that are retained by the acoustic field back to thebioreactor. The circulating flow motion is driven by the primaryrecirculation stream and can be optimized with acoustic chamber geometryvariations for maximum system efficiency. This will be discussed furtherbelow with respect to FIG. 33 below.

During perfusion, the acoustic perfusion devices of the presentdisclosure have multiple possible modes of operation. One of these modesmay be dominant in the device or they may occur concurrently dependingon the distribution of cells and fluid within the device. In a firstmode of operation illustrated in FIG. 41 (Mode 1), the fluid containingcells 4020 (light color) enters the acoustic standing wave field 4040,which is produced here between transducer 4008 and reflector 4009. Amulti-dimensional acoustic standing wave traps the cells at specificpoints, packs the cells into tightly packed clusters 4022, andcontinuously separates the clusters through enhanced gravitationalsettling. The cell clusters settle out, enter the tangential flow path(indicated by arrow 4001) and are redirected to the bioreactor by therecirculation stream. Smaller particles 4030 (darker color) are nottrapped by, and pass through, the acoustic standing wave, to beharvested. The harvest flow direction is indicated by arrow 4002. Theorientation of this device is significant, and the direction of gravityis also indicated.

The second mode of operation (Mode 2) is illustrated in FIG. 42, wherethe acoustophoretic system creates a strong barrier for cells andprevents cells from entering the acoustic field. Here, a barrier ofcells is established between the two fluids through the edge effect ofthe acoustic standing wave. A first clarified fluid stream 4050 containsthe smaller particles/desired byproducts 4030 within the acousticstanding wave field and the harvest stream. A second fluid stream 4055contains the retained cells 4020 upstream of the acoustic standing wavefield. The harvest flow direction is indicated by arrow 4002. In thismode of operation, an acoustic edge effect (indicated by dotted line4007) is realized. Very generally, the acoustic edge effect holds thecells back and prevents them from entering the acoustic field while aportion of the fluid stream containing the produced biomolecules andcell fragments is permitted to pass through the barrier created by theacoustic edge. The tangential flow path underneath the acoustic edge(arrow 4001) collects the retained cells and flows them back into themain recirculation stream and back to the bioreactor. This will also bediscussed further below with respect to FIG. 32. Again, the direction ofgravity is indicated.

In perfusion applications, the setup of the acoustophoretic device issimilar to that of TFF. A feed stream containing the cells, cell debris,fines, and product, i.e., protein, flows from the bioreactor into theperfusion system. A portion of the stream flows in a tangential fashionalong the lower edge of the acoustic standing wave and is recirculatedback to the bioreactor. A smaller portion of the feed stream isharvested, i.e., diverted and flows through the acoustic standing wave.Here the acoustic standing wave functions very similarly to the filterin TFF, preventing the cells from entering the acoustic field. Theharvest stream contains smaller particles such as cell debris and finesas well as the desired biomolecule product. The cells that are retainedby the acoustic standing wave are transported by the recirculationstream back to the bioreactor. FIG. 32, discussed further herein, alsoillustrates a perfusion device that uses a tangential flow stream.

Perfusion applications typically entail high cell densities, e.g., >50million cells/mL, and lower harvest velocities contrary to cellclarification or oil/water applications. The two fluid streams also havedifferent effective acoustic properties, i.e., speed of sound anddensity of the media/cell mixture. As cell density increases, thedifference in acoustic properties of the two fluid streams will be morepronounced as well. The acoustic standing wave field will now exert anacoustic radiation pressure, i.e., a pressure rise, on the second fluidstream, enriched with cells, as well as acoustic radiation forces on thecells suspended in the fluid. This radiation pressure and radiationforce act at the interface between the two fluids which coincides withthe upstream edge of the acoustic field. When this “acoustic edge”effect of acoustic radiation force is sufficiently strong, it willprevent the cells from entering the acoustic field. Equally important isa tangential flow path to collect the retained cells and transport themback to the bioreactor.

The acoustic edge effect may also be referred to as an acoustic walleffect and results from the edge of the acoustic field exerting a stronglateral force, i.e., in the direction of the harvest flow andperpendicular to the axis of the acoustic standing wave, on thesuspended particles, thereby keeping the relatively larger sizedparticles from entering the acoustic field and allowing only clarifiedfluid (i.e., the fluid containing the smaller-sized product) to enterthe acoustic field, thereby creating an acoustic perfusion cellretention device. In this way, only the clarified fluid can escape andthe cells are held down by the radiation force. This force is neverpositive, meaning that it always holds the cells down at the interface,i.e., the force is acting in the upstream flow direction, not allowingthem to escape the acoustic edge. The multiple peaks in the power curve(see discussion of FIG. 37 below) show the existence of multiple modesof operation including planar resonance modes and multi-dimensionalmodes of operation, indicating that this type of operation can begenerated through utilization of planar and multi-dimensional standingwaves alike. In systems having 1″×1″ dimensions, there exists a planarresonance about every 30 kHz. FIG. 37 shows evidence of additional peaksindicating the existence of the multi-dimensional modes. Per unit power,these modes can be equally or even more effective as the planarresonance modes. As explained above, the cells that are held back by theacoustic radiation force may then picked up by the scrubbing motion ofthe flow field (i.e., the recirculating flow underneath the interface),and be continuously returned to the bioreactor to ensure they receivethe nutrition and oxygen to maintain the production of the overall cellculture.

The clarified fluid contains both the desired products and cellfragments, all of which are smaller than whole viable cells. In thisway, the media that is returned to the bioreactor is clarified of cellfragments. Cell fragments absorb media without producing desiredproduct, making the perfusion process less efficient. Thus, there is anefficiency gain and a cost savings obtained by removing these cellfragments using the acoustic perfusion devices of the presentdisclosure. Further clarification of the clarified fluid may be achieveddownstream using a second device or a secondary flow chamber thatcontains another transducer-reflector pair that operates at a higherfrequency. This traps and agglomerates particles having a size of about10 microns or less that may have inadvertently passed through theoriginal acoustic standing wave, in the same manner as described before.Yet another transducer-reflector pair operating at still higherfrequencies, 3 MHz to 20 MHz, may be utilized to trap, agglomerate anddrop out the small cell fragments and debris that have passed throughthe initial acoustic standing wave and the “edge effect”. Thistriple-clarified fluid containing the desired biomolecules can thendirectly enter a sterile filter. For example, the original acousticperfusion device may operate at frequencies up to about 4 MHz. It iscontemplated that the frequency of this second and third acousticstanding wave field would be from about 6 MHz to about 20 MHz, andpossibly higher, to trap smaller sized cell fragments.

During startup of a bioreactor at low cell density, e.g., 2 millioncells/mL, the first described mode of operation dominates (FIG. 33, leftimage). As cell density in the bioreactor increases over time, the modeof operation gradually switches from mode 1 to mode 2, and both modesmay coexist at the same time.

When an acoustic standing wave is employed for perfusion in a bioreactorwith an already high cell density, e.g., 50 million cells/mL, the devicetypically starts in the first mode of operation (FIG. 33, left image),until the volume of fluid within the acoustic standing wave isclarified, at which point the operation gradually switches to the seconddescribed mode of operation (FIG. 33, right image). At times, duringoperation, an instability, usually manifested as a perturbation oroscillation of the interface between the two fluids, may growsufficiently strong such that cells enter the volume of fluid within theacoustic standing wave, at which point, for a short period of time, thedevice acts in a combined mode of operation, where both modes are active(i.e., the edge effect prevents cells from entering the acoustic fieldas explained above, while the acoustic field clarifies the cells thathave entered the volume of fluid within the acoustic standing wavefield). Once the tightly packed cell clusters have settled out (i.e.,once the volume of fluid within the acoustic standing wave has beensufficiently clarified), the mode of operation is then again that of thesecond described mode of operation including the acoustic edge effect.It is important to note that the device can operate in both/either ofthe modes of operation, as described above, without external switching.In other words, the properties of the fluid streams, e.g., cellconcentrations in the streams, and acoustic field dictate which modedominates.

The acoustic standing wave(s) perfusion devices of the presentdisclosure are operated differently compared to prior acoustic filterusages, previously described in literature. Previously, acoustophoresiswas operated such that the protein-producing materials, such as Chinesehamster ovary cells (CHO cells), the most common host for the industrialproduction of recombinant protein therapeutics, were trapped within theultrasonic standing wave (i.e., remain in a stationary position). Cellswere retained in an acoustic field by causing individual cells tomigrate towards the pressure nodal planes of the planar acousticstanding wave. There, as the cells were retained in the standing wave,there was also a physical scrubbing of the cell culture media flowingpast, whereby more cells were trapped as they came in contact with thecells that were already held within the standing wave. The standing wavewas then intermittently shut off to allow the cells to drop out of thestanding wave and return to the bioreactor.

In contrast, in the present disclosure, the ultrasonic standing wavesare used as a blanket or selector or “force field”. Rather than trappingthe biological cells within the standing wave, fluid flows through theperfusion device in a manner such that gravity first operates on thebiological cells, causing them to sink. The standing wave is creatednear the top of the filtering device and acts like a filter to preventthe cells from entering the acoustic field and exiting through the topof the filtering device (i.e., acting similar to a force field holdingthe cells back from entering the acoustic field). Thus, two outputstreams are created, one output stream retaining the cells and exitingthrough a port at the bottom of the device, and the other output streambeing depleted in cells and exiting through a port at the top of thedevice (the cell concentration in the two output streams being comparedto each other). There is almost no reliance on agglomeration of thecells within the acoustic field, which is particularly advantageous incertain applications because no retention time of the cells in theacoustic filtering device is required.

Described another way, the acoustic perfusion device has two fluidstreams flowing at different rates. The main fluid stream, carrying theexpressing cell culture, culture media, product, and other bioreactorconstituents, enters the device and is partially diverted into asecondary, lower volume, lower flow fluid stream. This secondary fluidstream passes through the multi-dimensional acoustic standing wave,where the multi-dimensional acoustic standing wave (or generally anacoustic standing wave) holds back the main cell culture and allows theexpressed biomolecules, the monoclonal antibodies and recombinantproteins, along with other small particles such as submicron andmicron-sized cell debris, to pass through and be further collected andprocessed outside/downstream of the bioreactor. The main fluid stream,containing the main cell culture, is then recycled back to thebioreactor.

In another application, the acoustic perfusion devices can act as aretention device and cell washing device for cell therapy applications.In continuous cell-culture applications, such as autologous andallogeneic cell therapy, it is necessary to purify, isolate, and thenproliferate cells that are initially harvested at a very lowcell-density. Relatively few cells seed a bioreactor, in which cellnumbers must be increased. Further processing steps such asconcentrating, washing, and media exchange are all needed for variousapplications. The commonality in all these applications is the need tocontinuously circulate, add, and/or remove media while retaining cellsin a bioreactor (which may be traditional or single-use) with no effectto their viability. The acoustic cell retention systems described hereinoperate over a range of cell recirculation rates, efficiently retaincells over a range of perfusion (or media removal rates), and can betuned to fully retain or selectively pass some percentage of cellsthrough power or frequency manipulation. Power and flow rates can all bemonitored and used as feedback in an automated control system. Specialtyflow paths may also be used such that a small volume of the main fluidflow is “sipped” off and the expressed biomolecules are separated fromthe main cell culture.

One advantage of acoustophoresis is that the acoustic radiation forcedoes not harm or negatively affect the biological cells or the desiredbiomolecule product. Moreover, perfusion is continuous, such that thecell culture is kept viable and desired products can be continuallyrecovered therefrom.

In a perfusion bioreactor system, it is desirable to be able to filterand separate the viable biological cells from the expressed materialsthat are in the fluid stream (i.e., cell culture media) and cellulardebris. As previously mentioned, such biological cells may includeChinese hamster ovary (CHO) cells, whose cell genome is manipulated toexpress large biomolecules. Such biomolecules can include recombinantproteins or monoclonal antibodies, and are the desired product to berecovered.

The acoustic perfusion devices of the present disclosure are designed tomaintain a high intensity multi-dimensional acoustic standing wave thatcan act as a filter, permitting smaller particles (such as recombinantproteins or cellular debris) to pass through while excluding largerparticles (such as viable cells). Generally, the device is driven by anoscillator and amplifier (not shown), and the device performance ismonitored and controlled by a computer (not shown). It may be necessary,at times, due to acoustic streaming, to modulate the frequency orvoltage amplitude of the standing wave. This may be done by amplitudemodulation and/or by frequency modulation. The duty cycle of thepropagation of the standing wave may also be utilized to achieve certainresults (i.e. the acoustic beam may be turned on and shut off atdifferent time periods or rates).

FIG. 1 illustrates a single standing wave system 100 that is comprisedof a reflector plate 101 and an ultrasonic transducer 103 that is set toresonate so as to form a standing wave 102. Excitation frequenciestypically in the range from 100 kHz to 100 MHz are applied by thetransducer 103. One or more multi-dimensional standing waves are createdbetween the transducer 103 and the reflector 101. An ideal standing waveis the sum of two propagating waves that are equal in frequency andintensity and that are traveling in opposite directions, i.e. from thetransducer to the reflector and back. The propagating wavesconstructively interfere with each other and thus generate the standingwave. A dotted line 105 is used to indicate the zero-amplitude of thewave. A node is a point where the wave has minimum amplitude, and isindicated with reference numeral 107. An anti-node is a point where thewave has maximum amplitude, and is indicated with reference numeral 109.

FIG. 2 is a schematic diagram that compares a fed-batch bioreactorsystem 201 (left side) with a perfusion bioreactor system 202 (rightside). Beginning with the fed-batch bioreactor on the left, thebioreactor 210 includes a reaction vessel 220. The cell culture media isfed to the reaction vessel through a feed inlet 222. An agitator 225 isused to circulate the media throughout the cell culture. Here, theagitator is depicted as a set of rotating blades, though any type ofsystem that causes circulation is contemplated. The bioreactor permitsgrowth of a seed culture through a growth/production cycle, during whichtime debris, waste and unusable cells will accumulate in the bioreactorand the desired product (e.g. biomolecules such as monoclonalantibodies, recombinant proteins, hormones, etc.) will be produced aswell. Due to this accumulation, the reaction vessel of a fed-batchprocess is typically much larger than that in a perfusion process. Thedesired product is then harvested at the end of the production cycle.The reaction vessel 220 also includes an outlet 224 for removingmaterial.

Turning now to the perfusion bioreactor 202 on the right-hand side,again, the bioreactor includes a reaction vessel 220 with a feed inlet222 for the cell culture media. An agitator 225 is used to circulate themedia throughout the cell culture. An outlet 224 of the reaction vesselis fluidly connected to the inlet 232 of an acoustic perfusion device230 of the present disclosure, and continuously feeds the bioreactorcontents (containing cells and desired product) to the filtering device.The perfusion device is located downstream of the reaction vessel, andseparates the desired product from the cells. The acoustic perfusiondevice 230 has two separate outlets, a product outlet 234 and a recycleoutlet 236. The product outlet 234 fluidly connects the acousticperfusion device 230 to a containment vessel 240 downstream of theperfusion device, which receives the flow of the desired product (plusmedia) from the perfusion device. From there, furtherprocessing/purification can occur to isolate/recover the desiredproduct. For example, further downstream of this acoustic perfusiondevice may be additional filters such as an ATF, TFF, depth filter,centrifuge, etc. The recycle outlet 236 fluidly connects the acousticperfusion device 230 back to a recycle inlet 226 of the reaction vessel220, and is used to send the cells and cell culture media back into thereaction vessel for continued growth/production. Put another way, thereis a fluid loop between the reaction vessel and the perfusion device.The reaction vessel 220 in the perfusion bioreactor system 202 has acontinuous throughput of product and thus can be made smaller. Thefiltering process is critical to the throughput of the perfusionbioreactor. A poor filtering process will allow for only low throughputand result in low yields of the desired product.

FIG. 3 is a cross-sectional view of a generic bioreactor 300 that isuseful for the systems of the present disclosure. As illustrated here,the bioreactor includes a reaction vessel 320 having an internal volume323. A feed inlet 322 at the top of the vessel is used to feed cellculture media into the vessel. An agitator 325 is present. An outlet 324is shown at the bottom of the vessel. A thermal jacket 310 surrounds thereaction vessel, and is used to regulate the temperature of thecells/media. An aerator 312 is located on the bottom of the vessel forproviding gas to the internal volume. Sensors 314 are shown at the topright of the vessel. A pump 316 is illustrated for feeding the cellculture media into the vessel, as is another pump 318 for removing cellculture media from the vessel.

The perfusion systems described above use an acoustic perfusion deviceof the present disclosure. The contents of the bioreactor arecontinuously flowed through the acoustic perfusion device to capture thedesired products.

FIG. 4 is a first embodiment of an acoustic perfusion device 400 thatcan be used with the previously-described systems. The device includesan inlet port 410, an outlet port 430, a first collection port 470, abottom wall 420, and an acoustic chamber 450. The acoustic chamber 450can also be referred to as a fluid cell.

The inlet port 410 is located at a first end 412 of the device.Generally, the inlet port 410 is fluidly connected to an associatedbioreactor and serves as the inlet through which the fluid medium withcells, fines, and product is introduced to the device. An inlet flowpath 451 leads from the inlet port 410 to the acoustic chamber 450,which contains an internal volume. An upper wall 411 can be presentabove the inlet flow path leading from the inlet port to the acousticchamber, the upper wall having a substantially horizontal orientation.The inlet flow path has a cross-sectional area 452 (illustrated by thedotted square).

The inlet port 410 is located at a first height 402 above the outletport 430, which defines a bottom end of the device. Put another way, theoutlet port 430 is located below the acoustic chamber 450 or below theinlet port 410, or at the bottom end 416 of the device. The placement ofthe outlet port 430 below the inlet port 410 ensures that fluid flowthrough the device is passively urged by gravity towards the outlet port430, and that a hydraulic head is created within the device. The outletport 430 may also be referred to as a fluid recycle port because thehost fluid is recycled or returned from the device to the associatedbioreactor through the outlet port 430. As illustrated here, the outletport 430 is also located at a second end 414 of the device, opposite thefirst end 412. The first end 412 and second end 414 can be considered asbeing opposite ends of an x-axis, while the bottom end 416 and top end418 can be considered as being opposite ends of a z-axis.

The first collection port 470 is located above the acoustic chamber 450at the top end 418 of the device, and is fluidly connected to theacoustic chamber. The device may include additional collection ports,such as second collection port 472, which is spaced apart from the firstcollection port 470. The first and second collection ports 470, 472 aregenerally used to harvest and recover a portion of the desiredbiomolecule byproducts from the device. A collection or harvest flowpath 453 leads from the acoustic chamber to the collection ports 470,472. The collection flow path has a cross-sectional area 454(illustrated by the dotted square). In some particular embodiments, thecross-sectional area 454 of the collection flow path is greater than thecross-sectional area 452 of the inlet flow path. This is one method bywhich the flow rate of fluid through the collection ports 470, 472 canbe made much lower than the incoming flow rate of fluid. When used inperfusion biomanufacturing, the collection ports can also be referred toas perfusion or harvest ports. Because fluid depleted in cells andenriched in desired biomolecule products, cell debris, and other finesis harvested, the collection ports can also be referred to as harvestports, and the collection flow path can also be referred to as theharvest flow path.

In this embodiment, the bottom wall 420 extends from the inlet port 410to the outlet port 430 of the device. The exact shape of the bottom wall420 can vary to obtain the desired fluid flow. As illustrated here, thebottom wall 420 curves from the inlet port 410 to the outlet port 430 ofthe device. Relative to a line between the inlet port 410 and the outletport 430, illustrated as dotted line 401, the bottom wall 420 has aconcave curve. An outlet flow path 432 leads from the acoustic chamber450 to the outlet port 430.

As illustrated here, a first ultrasonic transducer 460 is located on asidewall 440 of the device at a second height 404 that is above thefirst height 402 (i.e. closer to the top end 418 of the device) andbelow the collection ports 470, 472. This volume above the acousticchamber 450 and below the collection ports 470, 472 is identified hereas a harvest or collection zone 456. The first ultrasonic transducer 460includes a piezoelectric material that can be driven by a voltage signalto create a multi-dimensional standing wave in the acoustic chamber 450across the collection flow path 453. An acoustic radiation force fieldthus separates the acoustic chamber 450 from the collection ports 470,472.

In the embodiment of FIG. 4, the device includes a reflector 480 locatedon a wall opposite from the first ultrasonic transducer 460. Thereflector is also located at the second height (i.e. the same height asthe transducer). Together, the transducer 460 and reflector 480 generatea multi-dimensional acoustic standing wave, as illustrated in FIG. 1.

The inlet port 410, outlet port 430, and the collection ports 470, 472are, in this illustrated embodiment, all located on a front wall 475 ofthe device. It is also contemplated that these ports can face in anyother direction, as desired. The front wall 475 is illustrated here ashaving a flat or planar face, and has a constant thickness. However, theshape of the front wall may also vary if desired, for example to changethe cross-sectional areas 452, 454. Finally, the rear wall of the deviceis attached to a mounting piece 490, which contains holes 492 forattaching the perfusion device to a surface for operation.

In use, the fluid medium containing biological cells and smallermolecules enters the acoustic chamber 450 through the inlet port 410.Inside the acoustic chamber, gravity acts to drag the biological cellsdownwards towards the outlet port 430. A passive settling process occursin the acoustic chamber, creating a fluid with a relatively highconcentration of biological cells at the bottom end 416 of the device,and a fluid with a relatively lower concentration of biological cells atthe top end 418 of the device. The vast majority of incoming fluid, andthereby, the large majority of the cell population never passes throughthe acoustic standing wave(s). The fluid with the high concentration ofbiological cells is pumped back to the bioreactor, and the fluid withthe relatively low concentration of biological cells (and alsocontaining desired biomolecules) is pumped out and collected though thecollection port(s) 470, 472. The acoustic standing wave(s) of the deviceact to prevent significant numbers of biological cells from exitingthrough the collection port(s) 470, 472.

The flow rate through the collection or harvest flow path 453 is, invarious embodiments, at least one order of magnitude smaller than theflow rate through the inlet flow path 451. In more particularembodiments, the flow rate of the fluid medium entering the devicethrough the inlet port is about 1 liter per minute (L/min) and the flowrate of the fluid depleted in cells exiting the device through thecollection port(s) is about 10 milliliters per minute (mL/min). In sometests, bioreactors having a size of 2 liters to 10 liters have beentested with solutions containing up to 10% yeast and up to 50 millioncells/m L. The flow rate through the inlet port has been from about 0.75L/min to about 3 L/min, with the flow rate through the collection flowpath (i.e. all collection ports together) being about 1 mL/min to about30 mL/min. A 95% cell recovery rate has been achieved.

The acoustic perfusion devices of the present disclosure can filter veryhigh cell densities, around 100 million cells per mL and possibly in therange of about 60 million to about 120 million cells per mL, whereasother filtering technologies such as ATF can only filter at densitiesless than 80 million cells per mL. Unlike hollow fiber membranes, theacoustic standing wave(s) can also be tuned to allow passage of cells ifdesired, as well as allow the passage of fines/debris. This can act as acleaning operation for the bioreactor. Continuous, steady-stateoperation is possible without pressure fluctuations, and the productstream does not accumulate in the bioreactor or the filtering device.

The acoustic perfusion device can be made of appropriate materials knownin the art. Such materials include high density polyethylene (HDPE),other plastics, and potentially metals and glasses. It has been foundvery convenient for the device to be transparent, so that fluid flow andultrasonic transducer operation can be visually confirmed.

FIG. 5 shows another embodiment of an acoustic perfusion device 500.This embodiment is very similar to the device 400 depicted in FIG. 4.The main difference is that the acoustic perfusion device 500 of FIG. 5has a first ultrasonic transducer 460 on one sidewall of the device anda second ultrasonic transducer 562 on an opposite sidewall 440 thereofin the collection zone 456. Put another way, the two transducers 460,562 are located on opposite sides of the collection flow path 453. Withthis arrangement, the reflector 580 is located within the collectionzone 456 between the first and second ultrasonic transducers 460, 562.The transducers are oriented so that the reflector 580 and first andsecond ultrasonic transducers 460, 562 create multi-dimensional standingwave(s) in the fluid cell 450 as described above, or put another way thetransducers are facing each other. Also illustrated is the outflow pump592 attached to the outlet port 430 of the device, which is used tocontrol the flow rate of the fluid medium flowing through the device.Not illustrated here is the pump attached to the collection ports (notvisible) of the filtering device 500.

Turning now to FIG. 6, a processing system is shown including anassociated bioreactor 610 and an acoustic perfusion device 630 of thepresent disclosure. The system is set up for use as a perfusionbioreactor. The bioreactor 610 includes a reaction vessel 620 having afeed inlet 622, an outlet 624, and a recycle inlet 626. Fresh media isadded into the feed inlet 622 by an addition pipe 650. Some reactorswill also include an outlet or bleed port (not shown here) to remove or“bleed” cells in order to maintain a constant cell density within areactor. The contents of the reaction vessel (reference numeral 605) aremixed with an agitator 625. The desired product (e.g., recombinantproteins) is continuously produced by cells located within the vessel620, and are present in the media of the bioreactor. The product and thecells in the perfusion bioreactor are drawn from the reaction vesselthrough pipe 652, and enter the acoustic perfusion device 630 throughinlet port 632. Therein, a portion of the desired product is separatedfrom the cells. The desired product can be drawn off through a firstcollection port 634 (which is a product recovery port) and pipe 654 intoa containment vessel 640, or in the case of a truly continuousproduction system, some other downstream purification process. The cellsare returned to the perfusion bioreactor after separation, passing fromoutlet port 636 (which is a fluid recycle port) of the acousticperfusion device through pipe 656 to recycle inlet 626 of the reactionvessel, which form a recycle path. The multi-dimensional standingwave(s) of the acoustic perfusion device are used to create a separationbarrier between the fluid cell of the device and the collection port, sothat a highly reduced number of biological cells are collected incollection port 634.

FIG. 7 and FIG. 8 are views of another exemplary embodiment of anacoustic perfusion device. FIG. 7 is a front cross-sectional view, andFIG. 8 is an exterior perspective view. Notably, this embodiment isspecifically designed such that it can be fabricated with cleanmachining techniques, using Class VI materials (medical device gradeHDPE, for example), or even as single or welded injection molded part.In this manner, this embodiment is an example of a single-use device,which is gamma-stable. The devices are flushed to remove bioburden andthen gamma-irradiated (generally from 25-40 kGy) to sterilize anypotential contamination that could destroy a healthy cell culture, suchas that present in a perfusion bioreactor.

Referring first to FIG. 7, in this device 700, the inlet port 710 andthe collection port 770 are both located at the top end 718 of thedevice, or on the top wall 776 of the device. The outlet port 730 islocated at a bottom end 716 of the device. Here, the inlet port 710 andthe outlet port 730 are both on a first side 712 of the device. Theinlet flow path 751 is in the form of a channel 755 that runs from theinlet port downwards towards the bottom end and past the outlet port,the channel being separated from the acoustic chamber 750 (here, theseparation occurring by an internal wall 756). Fluid will flow downwardsin the channel, then rise upwards into the acoustic chamber 750. Thebottom wall 720 of the acoustic chamber is a sloped planar surface thatslopes down towards the outlet port 730. The location of the ultrasonictransducers 760 are shown here as two squares, between the top end andthe bottom end of the device. The collection flow path 753 is locatedabove the transducers.

Referring now to FIG. 8, the device 700 is shown as being formed withina three-dimensional rectangular housing 706. It can be seen that theoutlet port 730 at the bottom end 716 of the device is located on afront wall 775. Again, the collection port 770 and the inlet port 710are located on a top wall 776. A viewing window 708 made of atransparent material is present in the front wall. Through that viewingwindow, it can be seen that the ultrasonic transducers are mounted inthe rear wall 778 of the device housing. The viewing window acts as areflector to generate the multi-dimensional acoustic standing waves.

FIG. 9 and FIG. 10 are views of yet another exemplary embodiment of anacoustic perfusion device. FIG. 9 is a front cross-sectional view, andFIG. 10 is a perspective view.

Referring first to FIG. 9, in this device 900, there is an inlet port910 present on a front side 975 of the device along the first side 912of the device. An outlet port 930 (best seen in FIG. 10) is locateddirectly opposite and at the same height as the inlet port 910, and isalso located on first side 912. In this embodiment, there is a mainfluid stream that flows almost directly from the inlet port 910 to theoutlet port 930, and the inlet flow path 951 diverts only a small sideflow into the acoustic chamber 950 from the first side 912 of thedevice. The collection port 970 is located at the top end 918 of thedevice, or on the top wall 976 of the device. A secondary outlet port980 is located on the first side 912 of the device as well, extendingfrom first side wall 979, and located below the inlet port 910, and canact as a bleed port. The bottom wall 920 of the acoustic chamber isshaped in a pyramid-like fashion to taper downwards to a vertex. A drainline 981 runs from the bottom of the acoustic chamber 950 to thesecondary outlet port 980. It is contemplated that here, the secondaryoutlet port can be used to capture a small flow of highly concentratedcells, which can either be discarded (cell bleed) or can also bereturned back to the bioreactor.

Referring now to FIG. 10, the front wall 975 of the device has arectangular space 960, and the rear wall 978 of the device has arectangular space 962. It is contemplated that one transducer and onereflector can be placed in these two rectangular spaces 960/962 ineither orientation, or that two transducers could be placed in the tworectangular spaces. The inlet port 910 and outlet port 930 are bothvisible in this view. The inlet port 910 is located on the front side ofthe device, and the outlet port 930 is located on the rear side of thedevice (though this could be reversed if desired). The clarificationflow path 953 is located above the transducers. Although not depictedhere, a mounting piece similar to that in FIG. 4 could be attached tothe second side 914 of the device.

It may be helpful now to describe the ultrasonic transducer(s) used inthe acoustic filtering device in more detail. FIG. 11 is across-sectional diagram of a conventional ultrasonic transducer. Thistransducer has a wear plate 50 at a bottom end, epoxy layer 52, ceramicpiezoelectric element 54 (made of, e.g. Lead Zirconate Titanate (PZT)),an epoxy layer 56, and a backing layer 58. On either side of the ceramicpiezoelectric element, there is an electrode: a positive electrode 61and a negative electrode 63. The epoxy layer 56 attaches backing layer58 to the piezoelectric element 54. The entire assembly is contained ina housing 60 which may be made out of, for example, aluminum. Thehousing is used as the ground electrode. An electrical adapter 62provides connection for wires to pass through the housing and connect toleads (not shown) which attach to the piezoelectric element 54.Typically, backing layers are designed to add damping and to create abroadband transducer with uniform displacement across a wide range offrequency and are designed to suppress excitation of particularvibrational eigen-modes of the piezoelectric element. Wear plates areusually designed as impedance transformers to better match thecharacteristic impedance of the medium into which the transducerradiates.

FIG. 12 is a cross-sectional view of an ultrasonic transducer 81 of thepresent disclosure, which is used in the acoustic filtering device ofthe present disclosure. Transducer 81 is shaped as a square, and has analuminum housing 82. The aluminum housing has a top end and a bottomend. The transducer housing may also be composed of plastics, such asmedical grade HDPE or other metals. The piezoelectric element is a massof perovskite ceramic, each consisting of a small, tetravalent metalion, usually titanium or zirconium, in a lattice of larger, divalentmetal ions, usually lead or barium, and O²⁻ ions. As an example, a PZT(lead zirconate titanate) piezoelectric element 86 defines the bottomend of the transducer, and is exposed from the exterior of the bottomend of the housing. The piezoelectric element is supported on itsperimeter by a small elastic layer 98, e.g. epoxy, silicone or similarmaterial, located between the piezoelectric element and the housing. Putanother way, no wear plate or backing material is present. However, insome embodiments, there is a layer of plastic or other materialseparating the piezoelectric element from the fluid in which theacoustic standing wave is being generated. The piezoelectricelement/crystal has an exterior surface (which is exposed) and aninterior surface as well.

Screws 88 attach an aluminum top plate 82 a of the housing to the body82 b of the housing via threads. The top plate includes a connector 84for powering the transducer. The top surface of the PZT piezoelectricelement 86 is connected to a positive electrode 90 and a negativeelectrode 92, which are separated by an insulating material 94. Theelectrodes can be made from any conductive material, such as silver ornickel. Electrical power is provided to the PZT piezoelectric element 86through the electrodes on the piezoelectric element. Note that thepiezoelectric element 86 has no backing layer or epoxy layer. Putanother way, there is an interior volume or an air gap 87 in thetransducer between aluminum top plate 82 a and the piezoelectric element86 (i.e. the air gap is completely empty). A minimal backing 58 and/orwear plate 50 may be provided in some embodiments, as seen in FIG. 13.

The transducer design can affect performance of the system. A typicaltransducer is a layered structure with the ceramic piezoelectric elementbonded to a backing layer and a wear plate. Because the transducer isloaded with the high mechanical impedance presented by the standingwave, the traditional design guidelines for wear plates, e.g., halfwavelength thickness for standing wave applications or quarterwavelength thickness for radiation applications, and manufacturingmethods may not be appropriate. Rather, in one embodiment of the presentdisclosure the transducers, there is no wear plate or backing, allowingthe piezoelectric element to vibrate in one of its eigenmodes with ahigh Q-factor, or in a combination of several eigenmodes. The vibratingceramic piezoelectric element/disk is directly exposed to the fluidflowing through the fluid cell.

Removing the backing (e.g. making the piezoelectric element air backed)also permits the ceramic piezoelectric element to vibrate at higherorder modes of vibration with little damping (e.g. higher order modaldisplacement). In a transducer having a piezoelectric element with abacking, the piezoelectric element vibrates with a more uniformdisplacement, like a piston. Removing the backing allows thepiezoelectric element to vibrate in a non-uniform displacement mode. Thehigher order the mode shape of the piezoelectric element, the more nodallines the piezoelectric element has. The higher order modal displacementof the piezoelectric element creates more trapping lines, although thecorrelation of trapping line to node is not necessarily one to one, anddriving the piezoelectric element at a higher frequency will notnecessarily produce more trapping lines.

It is contemplated that, in some embodiments of the acoustic filteringdevice of the present disclosure, the piezoelectric element may have abacking that minimally affects the Q-factor of the piezoelectric element(e.g. less than 5%). The backing may be made of a substantiallyacoustically transparent material such as balsa wood, foam, or corkwhich allows the piezoelectric element to vibrate in a higher order modeshape and maintains a high Q-factor while still providing somemechanical support for the piezoelectric element. The backing layer maybe a solid, or may be a lattice having holes through the layer, suchthat the lattice follows the nodes of the vibrating piezoelectricelement in a particular higher order vibration mode, providing supportat node locations while allowing the rest of the piezoelectric elementto vibrate freely. The goal of the lattice work or acousticallytransparent material is to provide support without lowering the Q-factorof the piezoelectric element or interfering with the excitation of aparticular mode shape.

Placing the piezoelectric element in direct contact with the fluid alsocontributes to the high Q-factor by avoiding the dampening and energyabsorption effects of the epoxy layer and the wear plate. Otherembodiments of the transducer(s) may have wear plates or a wear surfaceto prevent the PZT, which contains lead, contacting the host fluid. Thismay be desirable in, for example, biological applications such asseparating blood, biopharmaceutical perfusion, or fed-batch filtrationof mammalian cells. Such applications might use a wear layer such aschrome, electrolytic nickel, or electroless nickel. Chemical vapordeposition could also be used to apply a layer of poly(p-xylylene) (e.g.Parylene) or other polymer. Organic and biocompatible coatings such assilicone or polyurethane are also usable as a wear surface.

In some embodiments, for applications such as oil/water emulsionsplitting and others such as perfusion, the ultrasonic transducer has anominal 2 MHz resonance frequency. Each transducer can consume about 28W of power for droplet trapping at a flow rate of 3 GPM. This translatesto an energy cost of 0.25 kW hr/m³. This is an indication of the verylow cost of energy of this technology. Desirably, each transducer ispowered and controlled by its own amplifier. In other embodiments, theultrasonic transducer uses a square piezoelectric element, for examplewith 1″×1″ dimensions. Alternatively, the ultrasonic transducer can usea rectangular piezoelectric element, for example with 1″×2.5″dimensions. Power dissipation per transducer was 10 W per 1″×1″transducer cross-sectional area and per inch of acoustic standing wavespan in order to get sufficient acoustic trapping forces. For a 4″ spanof an intermediate scale system, each 1″×1″ square transducer consumes40 W. The larger 1″×2.5″ rectangular transducer uses 100 W in anintermediate scale system. The array of three 1″×1″ square transducerswould consume a total of 120 W and the array of two 1″×2.5″ transducerswould consume about 200 W. Arrays of closely spaced transducersrepresent alternate potential embodiments of the technology. Transducersize, shape, number, and location can be varied as desired to generatedesired multi-dimensional acoustic standing wave patterns.

The size, shape, and thickness of the transducer determine thetransducer displacement at different frequencies of excitation, which inturn affects separation efficiency. Typically, the transducer isoperated at frequencies near the thickness resonance frequency (halfwavelength). Gradients in transducer displacement typically result inmore trapping locations for the cells/biomolecules. Higher order modaldisplacements generate three-dimensional acoustic standing waves withstrong gradients in the acoustic field in all directions, therebycreating equally strong acoustic radiation forces in all directions,leading to multiple trapping lines, where the number of trapping linescorrelate with the particular mode shape of the transducer.

To investigate the effect of the transducer displacement profile onacoustic trapping force and separation efficiencies, an experiment wasrepeated ten times using a 1″×1″ square transducer, with all conditionsidentical except for the excitation frequency. Ten consecutive acousticresonance frequencies, indicated by circled numbers 1-9 and letter A onFIG. 14, were used as excitation frequencies. The conditions wereexperiment duration of 30 min, a 1000 ppm oil concentration ofapproximately 5-micron SAE-30 oil droplets, a flow rate of 500 ml/min,and an applied power of 20 W. Oil droplets were used because oil is lessdense than water, and can be separated from water using acoustophoresis.

FIG. 14 shows the measured electrical impedance amplitude of a squaretransducer as a function of frequency in the vicinity of the 2.2 MHztransducer resonance. The minima in the transducer electrical impedancecorrespond to acoustic resonances of the water column and representpotential frequencies for operation. Additional resonances exist atother frequencies where multi-dimensional standing waves are excited.Numerical modeling has indicated that the transducer displacementprofile varies significantly at these acoustic resonance frequencies,and thereby directly affects the acoustic standing wave and resultingtrapping force. Since the transducer operates near its thicknessresonance, the displacements of the electrode surfaces are essentiallyout of phase. The typical displacement of the transducer electrodes isnot uniform and varies depending on frequency of excitation. As anexample, at one frequency of excitation with a single line of trappedoil droplets, the displacement has a single maximum in the middle of theelectrode and minima near the transducer edges. At another excitationfrequency, the transducer profile has multiple maxima leading tomultiple trapped lines of oil droplets. Higher order transducerdisplacement patterns result in higher trapping forces and multiplestable trapping lines for the captured oil droplets.

As the oil-water emulsion passed by the transducer, the trapping linesof oil droplets were observed and characterized. The characterizationinvolved the observation and pattern of the number of trapping linesacross the fluid channel, as shown in FIG. 15, for seven of the tenresonance frequencies identified in FIG. 14. Different displacementprofiles of the transducer can produce different (more) trapping linesin the standing waves, with more gradients in displacement profilegenerally creating higher trapping forces and more trapping lines.

FIG. 16 is a numerical model showing a pressure field that matches the 9trapping line pattern. The numerical model is a two-dimensional model;and therefore only three trapping lines are observed. Two more sets ofthree trapping lines exist in the third dimension perpendicular to theplane of the page.

The lateral force of the acoustic radiation force generated by thetransducer can be increased by driving the transducer in higher ordermode shapes, as opposed to a form of vibration where the crystaleffectively moves as a piston having a uniform displacement. Theacoustic pressure is proportional to the driving voltage of thetransducer. The electrical power is proportional to the square of thevoltage. The transducer is typically a thin piezoelectric plate, withelectric field in the z-axis and primary displacement in the z-axis. Thetransducer is typically coupled on one side by air (i.e., the air gapwithin the transducer) and on the other side by the fluid medium of thecell culture media. The types of waves generated in the plate are knownas composite waves. A subset of composite waves in the piezoelectricplate is similar to leaky symmetric (also referred to as compressionalor extensional) Lamb waves. The piezoelectric nature of the platetypically results in the excitation of symmetric Lamb waves. The wavesare leaky because they radiate into the water layer, which result in thegeneration of the acoustic standing waves in the water layer. Lamb wavesexist in thin plates of infinite extent with stress free conditions onits surfaces. Because the transducers of this embodiment are finite innature, the actual modal displacements are more complicated.

FIG. 17 shows the typical variation of the in-plane displacement(x-displacement) and out-of-plane displacement (y-displacement) acrossthe thickness of the plate, the in-plane displacement being an evenfunction across the thickness of the plate and the out-of-planedisplacement being an odd function. Because of the finite size of theplate, the displacement components vary across the width and length ofthe plate. In general, a (m,n) mode is a displacement mode of thetransducer in which there are m undulations in transducer displacementin the width direction and n undulations in the length direction, andwith the thickness variation as described in FIG. 17. The maximum numberof m and n is a function of the dimension of the crystal and thefrequency of excitation. Additional three-dimensional modes exist thatare not of the form (m,n).

The transducers are driven so that the piezoelectric element vibrates inhigher order modes of the general formula (m, n), where m and n areindependently 1 or greater. Generally, the transducers will vibrate inhigher order modes than (2,2). Higher order modes will produce morenodes and antinodes, result in three-dimensional standing waves in thewater layer, characterized by strong gradients in the acoustic field inall directions, not only in the direction of the standing waves, butalso in the lateral directions. As a consequence, the acoustic gradientsresult in stronger trapping forces in the lateral direction.

In embodiments, the voltage signal driving the transducer can have asinusoidal, square, sawtooth, pulsed, or triangle waveform; and have afrequency of 500 kHz to 10 MHz. The voltage signal can be driven withpulse width modulation, which produces any desired waveform. The voltagesignal can also have amplitude or frequency modulation start/stopcapability to eliminate streaming.

The transducers are used to create a pressure field that generatesacoustic radiation forces of the same order of magnitude both orthogonalto the standing wave direction and in the standing wave direction. Whenthe forces are roughly the same order of magnitude, particles of size0.1 microns to 300 microns will be moved more effectively towards“trapping lines”, so that the particles will not pass through thepressure field and continue to exit through the collection ports of thefiltering device. Instead, the particles will remain within the acousticchamber to be recycled back to the bioreactor.

The following examples are provided to illustrate the devices,components, and methods of the present disclosure. The examples aremerely illustrative and are not intended to limit the disclosure to thematerials, conditions, or process parameters set forth therein.

EXAMPLES Example 1

FIG. 18 shows an experimental setup for an acoustic perfusion device asdescribed in detail above. This acoustic perfusion device is verysimilar to that illustrated in FIG. 5, except the bottom wall is notcurved, but rather runs horizontally from the first end and then anglesdirectly to the outlet port. Tubes are connected to the inlet port,outlet port, and the two collection ports. A pump is also visiblyfluidly connected to the outlet port.

FIG. 19 is a picture of another acoustic perfusion device of the presentdisclosure, similar to the embodiment shown in FIG. 5, having twoultrasonic transducers and a concave bottom wall leading from the inletport to the outlet port at the bottom end of the device. A cellcontaining fluid medium is also present in the device. In this picture,acoustic standing waves are created in the collection zone between thereflector and first and second transducers as described above. Theacoustic field generated thereby is indicated by waves and referencenumeral 1664. The flow pattern of the fluid medium through the devicefrom the inlet port to the outlet port is shown with an arrow (referencenumeral 1610) indicating the direction of fluid flow into the device andarrows (reference numeral 1630) indicating the direction of fluid flowthrough the device towards the outlet port. Finally, the general flowpattern of the desired product out of the device through the first andsecond collection ports is shown with arrows (reference numeral 1670)indicating the direction of flow.

Acoustophoretic separation has been tested using the acoustic perfusiondevice of FIG. 19 and the methods of separation of the presentdisclosure on different lines of Chinese hamster ovary (CHO) cells.FIGS. 20-28 show various test results varying different parameters andmeasuring different values using a Beckman Coulter Cell ViabilityAnalyzer.

The perfusion flow rates with the acoustic filtering device were fromabout 2 mL/min to about 10 mL/min, or the flow rates were about 1 VVD toabout 5 VVD for a 2.7 L working volume bioreactor. The VVD refers to the“vessel volume per day”, or how many times the volume of the bioreactorvessel is cycled through the acoustic filtering device in one day. Theperfusion flow rate (Qp) was collected through the perfusion ports. Incontrast, the feed flow rates (Qf) were from about 40 mL/min to about200 mL/min.

The feed solution had a starting CHO cell density of 50×10⁶ cells/mL.The reactor size was 2.7 L and the feed volume of the host fluid was 1.5L. In total, a series of seven tests (T1-T7) were performed to study theeffect of varying the VVD and flow split in a 2.7 L volume reactor. Theparameters for the tests are shown in Table 1 below.

TABLE 1 System results for a 2.7 L reactor and feed volume of 1.5 L FlowSplit Qp Qf (Qp/Qf) 1 VVD 1.5 VVD 2 VVD 5.2 VVD 5.0% T1 T2 T3 T7 2.5% T4T5 T6

The results included a cell clarification efficiency between 89-93% at aDC voltage of 45V, regardless of the flow rate as shown in FIG. 20. Itis noted that the DC voltage for T1 was fixed at 60V, whereas for testsT2-T7 the DC voltage was reduced to a fixed amount of 45V. Thetransducer voltage amplitude is half of these values.

The results further included a perfusate turbidity reduction of 90-94%compared to the feed, as shown in FIG. 21. This figure shows theturbidity of the feed, the recirculated fluid (Qc), and the perfusate(Qp). The feed entered the inlet port, the recirculated fluid exited theoutlet port and was recirculated, and the perfusate exited the perfusionport of the acoustic filtering device. It is noted that the turbiditymeasurements for tests T1 and T2 resulted in a hardware error, so onlytests T3-T7 are displayed, which equated to a 6-10% turbidity in theperfusion stream relative to the feed stream, regardless of flow rate.

FIG. 22 was produced by a Beckman Coulter Cell Viability Analyzer andrevealed a cell viability for each flow rate that was within the errorof instrument (i.e., ±6%), with the control ranging from 79-84% over alltests.

Further testing was performed using a solution designated “CHO Line A”.The solution had a starting cell density of 50×10⁶ cells/mL, a turbidityof 2,400 NTU, and cell viability of roughly 80%. The solution wasseparated using a device of the present disclosure in a system having areactor size of 2.7 L. The volume of the feed fluid was between 1.5 Land 2.0 L. The perfused flow rates were from 2 mL/min to 10 mL/min, orfrom 1 to 5 VVD. A series of six tests were performed to study theeffect of varying the VVD and flow split on acoustic filtrationperformance for the 2.7 L volume reactor. The parameters for the testsare shown in Table 2 below.

TABLE 2 System results for a 2.7 L reactor and feed volume from 1.5L-2.0 L T1 T2 T3 VVD 1.5 VVD 2 VVD 1 Flow Split 5.00% Flow Split 5.00%Flow Split 2.50% Perfused 2.8 Perfused 3.8 Perfused 1.9 Flow Flow Flow(ml/min) (ml/min) (ml/min) Feed Flow 56 Feed Flow 75 Feed Flow 75(ml/min) (ml/min) (ml/min) T4 T5 T6 VVD 1.5 VVD 2 VVD 5.2 Flow Split2.50% Flow Split 2.50% Flow Split 5.00% Perfused 2.8 Perfused 3.8Perfused 10 Flow Flow Flow (ml/min) (ml/min) (ml/min) Feed Flow 112.5Feed Flow 150 Feed Flow 194.2 (ml/min) (ml/min) (ml/min)

FIG. 23 shows the measured total cell density of feed flow,recirculation flow, and perfused flow. The bioreactor cell retention forthe tests shows an approximately 90% perfusion efficiency. FIG. 24 showsthe measured cell viability for the tests, revealing no significantchange in viability across the tests.

Next, additional testing was performed using a solution designated “CHOLine B”. The solution had a starting cell density of 75×10⁶ cells/mL, aturbidity of 2,300 NTU, and cell viability of roughly 80%. The solutionwas separated using a device of the present disclosure in a systemhaving a reactor size of 2.7 L. Four tests were performed (T1-T4). Twoof the tests (T1, T3) used a device having a single transducer. Theother two tests (T2, T4) used a device having two transducers in series(such that the fluid ran through both standing waves). The parametersfor the tests are shown in Table 3 below.

TABLE 3 System results for a 2.7 L reactor and feed volume from 1.5L-2.0 L T1 T2 Transducers 1 Transducers 2 VVD 1 VVD 1 Perfused Flow(mL/min) 1.9 Perfused Flow (mL/min) 1.9 Feed Flow (mL/min) 75 Feed Flow(mL/min) 75 T3 T4 Transducers 1 Transducers 2 VVD 2 VVD 2 Perfused Flow(mL/min) 3.8 Perfused Flow (mL/min) 3.8 Feed Flow (mL/min) 150 Feed Flow(mL/min) 150

FIG. 25 shows the measured total cell density of feed, recirculation,and perfusion flows. The bioreactor cell retention for the tests shows aperfusion efficiency greater than 90%. The results further evidenced anapproximately 3-5% greater efficiency when using two transducers ratherthan a single transducer. FIG. 26 shows the measured cell viability forthe tests, revealing no significant change in viability across thetests. Practically speaking, operating at low VVD offers a number ofadvantages, such as media cost reduction.

In biological applications, it is contemplated that all of the parts ofthe system (i.e., the bioreactor, acoustic filtering device, tubingfluidly connecting the same, etc.) can be separated from each other andbe disposable. Avoiding centrifuges and filters allows better separationof the CHO cells without lowering the viability of the cells. Thetransducers may also be driven to create rapid pressure changes toprevent or clear blockages due to agglomeration of CHO cells. Thefrequency of the transducers may also be varied to obtain optimaleffectiveness for a given power.

Example 2

FIG. 27 shows another experimental setup for an acoustic perfusiondevice similar to that illustrated in FIG. 8. Tubes are connected to theinlet port, outlet port, and the collection port.

The device was tested at a transducer voltage of 40V peak to peak, aperfused flow rate of 15-30 mL/min, and a recirculation flow rate of 2L/min. Samples were taken every 45-60 minutes, and the cell retentionrate was determined. FIG. 28 shows the results. The y-axis is theretention, stated in terms of percentage (calculated by comparing theoutput cell count with the input, or bioreactor/culture cell count). Thex-axis is both the applied DC voltage (in V) and the perfused, orharvest, flow rate (in mL/min); it is merely coincidental that the rangeof numeric values for V and mL/min are the same. The cell retentionefficiency remained above 95% for perfused flow rates up to 20 mL/min,and remained above 90% up to about 25 mL/min. FIG. 33 is a compositepicture showing the device in a startup or cell settling mode (left) andin a steady state cell retention mode (right).

Next, experiments were performed to determine what factors would affectcell retention. The perfused flow rate was varied, as was the transducervoltage. When the perfused flow rate was varied, the transducer voltagewas maintained at 40V peak to peak and the recirculation flow rate wasmaintained at 2 L/min. When the transducer voltage was varied, theperfused flow rate was maintained at 20 mL/min and the recirculationflow rate was maintained at 2 L/min. The results indicated that, forthis particular embodiment, a perfused flow rate of about 15 mL/min toabout 28 mL/min was optimum, and a transducer voltage of about 15V peakto peak to about 28V peak to peak was optimum.

A better understanding of the added functionality provided by anacoustic perfusion device can be demonstrated by examining the observedcell samples coming in and being harvested from the device. FIG. 29A isa microscope image (from a Vi-Cell cell counter) of the feed suspension,here a viable cell culture population with approximately 56 millioncells/mL. Several large, round, healthy cells can be observed. FIG. 29Bis a histogram showing the distribution of cell diameter in thepopulation. The diameter distribution is strongly bi-modal, aroundvalues of about 11 microns and about 23 microns. These two modescorrespond roughly with the smaller debris and non-viable cells, and thelarger viable cells. It should be noted that this sample is from aparticularly “dirty” cell population. In general, a production cell linewould be far cleaner, and the peak at approximately 11 microns would bemuch smaller, or even non-existent.

FIG. 30A is another microscope image (from a Vi-Cell cell counter), thistime of the flow harvested from the acoustic perfusion device. In thisimage, very few bright, large cells are observed, in contrast to FIG.29A. Rather, the image is filled with more smaller, darker particles, ordebris. The experimental conditions in this case were a perfused rate of4 mL/min and a recirculation rate of 2 L/min with a DC input voltage of30V. This qualitative observation is confirmed by the histogram in FIG.30B, which shows the distribution of diameters in the perfusate. Lookingat FIG. 30B, the particle distribution is now unimodal, with the peak ofapproximately 9 microns. This indicates that the larger, viable cellshave been trapped and retained, or otherwise largely prevented fromexiting in the perfusate. Only smaller cells are passing through,together with cell debris, fines, and fragments, from submicron tomicron sized.

A computational fluid dynamics (CFD) model was made of this device. FIG.31 shows the velocity distributions within the device after 500 seconds.The units are in meters/second (m/s). As expected, the highestvelocities are found in the channel leading downwards from the inletport to the outlet port. The velocity is near zero in the fluid cell andout through the collection port. This is important for two reasons: theacoustic field is more effective in a flow with a lower, more uniformvelocity, and because the cells used in bio-manufacturing are sensitiveto flow, and the induced shear rate.

FIG. 32 is a diagram illustrating several aspects of this embodiment.Fluid flows into the device through the inlet port 710 (arrow 780) andinto the acoustic chamber, above the acoustic edge 783. The volume offluid 750 below the acoustic chamber contains the tangential flow path,indicated by arrow 782. Fluid with a relatively high amount of viablecells will exit through the outlet port 730, as indicated by arrow 781.The acoustic edge effect created by the standing waves is marked withreference numeral 783, and separates large cells from smaller cellfragments, particulate debris, desired biomolecules, etc., which canpass through the acoustic standing wave field 784. This harvest flowstream 785 then exits through harvest port 770. The tangential flow pathis part of the inlet flow path, and is located below the acoustic edge783 generated by the acoustic standing wave. The tangential flow pathwill transport away both the clusters of cells that drop from theacoustic standing wave field 784 due to gravity effects and the cellsthat are retained by the acoustic edge effect.

Example 3

Another way of explaining the operation of the acoustic perfusion devicecan be understood by looking at the results of a numerical study. In thenumerical study, two fluids with differing effective acoustic properties(i.e., speed of sound and density), were modeled with an interfacebetween them in COMSOL, a numerical simulation software. The acousticfield is calculated and therefrom the lateral radiation force acting ona particle in the direction of the fluid velocity is calculated usingGorkov's equation.

FIG. 34 shows the geometry of the simulation, utilizing a piezoelectrictransducer, steel reflector, aluminum housing, and two fluids: the firstfluid being water within the acoustic field and the second fluid being a15% concentration of CHO cells in water solution outside of the acousticfield, the second fluid having a higher density and higher speed ofsound than the water fluid.

The two fluids were separated as indicated by the solid line in themodel of FIG. 34. In this setup, the fluid velocity through the systemwas in a horizontal direction from left to right. Therefore, in order toact as a retention device, the acoustic field needs to generate a forceon the cells that acts in the negative x-direction (i.e., opposite thefluid velocity). Water was modeled with a fluid density of 1000 kg/m³and a speed of sound of 1500 m/s. CHO cells were modeled having adensity of 1050 kg/m³ and a speed of sound of 1550 m/s. A coupledmulti-physics numerical simulation that included a full piezoelectricsimulation of the piezoelectric material, an acoustic simulation of thetwo fluids, and a linear elastic simulation in the steel and aluminumbodies was performed at various frequencies of excitation. Thetransducer was driven at a peak voltage of 40 V.

FIGS. 35A-35C show the acoustic pressure in the two fluids and thedisplacement of the piezoelectric material, the aluminum housing, andthe steel reflector of the model at frequencies of operation of 2.218MHz, 2.2465 MHz, and 2.3055 MHz. The lateral radiation force (i.e.,horizontally in the direction of the fluid flow), was calculated at theinterface between the two fluids along with real electrical powerconsumed by the transducer. The structural displacement of thetransducer and steel are shown, along with the acoustic pressure in thefluid.

FIG. 36 shows the lateral radiation force (N) and the lateral radiationforce normalized by power (N/W) versus frequency acting on the suspendedCHO cells. This graph shows that at the resonance frequencies (i.e.,local maxima in power), the average lateral radiation force on theinterface is negative, meaning that it is in the negative-x direction.The result is the creation of an acoustic wall effect or an acousticedge effect. That is, the edge of the acoustic field exerts a stronglateral force on the suspended particles, thereby keeping the largerparticles from entering the acoustic field and allowing only the firstfluid (i.e., fluid containing only smaller particles, such as thedesired product, and excluding whole cells) to enter the acoustic field,thereby creating an acoustic perfusion cell retention device. In thisway, only the clarified fluid can escape and the cells are held down bythe radiation force. This force is never positive, meaning that italways holds the cells down at the interface, not allowing them toescape the acoustic edge. The multiple peaks in the power curve show theexistence of multiple modes of operation including planar resonancemodes and multi-dimensional modes of operation, indicating that thistype of operation can be generated through utilization of planar andmulti-dimensional standing waves alike. In systems having 1″×1″dimensions, there exists planar resonance about every 30 kHz. The graphshows evidence of additional peaks indicating the existence of themulti-dimensional modes. Per unit power, these modes can be equally oreven more effective as the planar resonance modes. As explained above,the cells that are held back by the acoustic radiation force may then bepicked up by the scrubbing motion of the flow field (i.e., therecirculating flow underneath the interface), and be continuouslyreturned to the bioreactor to ensure they receive the nutrition andoxygen to maintain the production of the overall cell culture.

Example 4

FIG. 37 and FIG. 38 show another experimental setup for an acousticperfusion device similar to that illustrated in FIG. 9. Tubes areconnected to the inlet port, outlet port, the collection port, and thesecondary outlet port (for a flow concentrated cells). Arrows areincluded to illustrate fluid flow. Arrows indicate the flow into theinlet port; the flow out of the outlet port; the perfusate flow out thetop of the device and the flow of concentrate out the bottom of thedevice. The flow through the inlet port to the outlet port is therecirculation flowrate. The perfusate flow out the top of the device isthe perfused flowrate containing clarified fluid depleted in cells andcontaining desired product. The flow of concentrate out the bottom ofthe device is the concentrated cell flow. The concentrated cell flow canbe used for a cell bleed operation or if desired, the cells can bereturned to the bioreactor.

The device was tested at a transducer voltage of 40V peak to peak, aperfused flow rate (out the top) of 1-10 mL/min, a recirculation flowrate of 0.75-1 L/min, and a concentrate flow rate (out the bottom) of 15mL/min. The cell retention rate was determined for different perfusedflowrates. FIG. 39 shows the results. The y-axis is the retention with1.00 indicating 100% retention. The cell retention efficiency remainedabove 98% for perfused flow rates up to 7 mL/min, and was just below 90%at 10 mL/min.

The present disclosure has been described with reference to exemplaryembodiments. Obviously, modifications and alterations will occur toothers upon reading and understanding the preceding detaileddescription. It is intended that the present disclosure be construed asincluding all such modifications and alterations insofar as they comewithin the scope of the appended claims or the equivalents thereof.

1. An acoustic perfusion device, comprising: an acoustic chamber; aninlet port, an inlet flow path leading from the inlet port to theacoustic chamber; an outlet port for recirculating a fluid/cell mixture;at least one collection port for collecting harvest fluid from thefluid/cell mixture; and at least one ultrasonic transducer and at leastone reflector opposite the at least one ultrasonic transducer, whereinthe at least one ultrasonic transducer includes a piezoelectric materialdriven by a voltage signal to create a multi-dimensional acousticstanding wave across a collection flow path leading from the acousticchamber to the at least one collection port.
 2. The device of claim 1,wherein the acoustic chamber is below the at least one collection port.3. The device of claim 1, wherein the inlet flow path is shaped togenerate a tangential flow path below an acoustic field generated by theacoustic standing wave.
 4. The device of claim 1, wherein a pressurerise and an acoustic radiation force on cells are generated at an edgeof the acoustic standing wave to clarify fluid passing through theacoustic standing wave.
 5. The device of claim 1, wherein the at leastone ultrasonic transducer is mounted in a rear wall or a front wall ofthe device.
 6. The device of claim 1, wherein the at least one reflectoris made of a transparent material.
 7. The device of claim 1, wherein theoutlet port is below the inlet port.
 8. The device of claim 1, having atotal of two or more ultrasonic transducers located on multiple sides ofthe collection flow path.
 9. The device of claim 8, where the at leastone reflector is located between the two or more ultrasonic transducers.10. The device of claim 8, wherein the two or more ultrasonictransducers are within the collection flow path and the at least onereflector is opposite the two or more ultrasonic transducers in a wallof the device.
 11. The device of claim 1, wherein the at least onetransducer is an array.
 12. The device of claim 1, wherein the acousticstanding wave results in an acoustic radiation force having an axialforce component and a lateral force component that are of the same orderof magnitude.
 13. The device of claim 1, having two or more collectionports spaced apart from each other on the top end of the device.
 14. Thedevice of claim 1, further comprising a secondary flow chamber in whichthe harvest fluid passes through a second acoustic standing wave havinga frequency higher than the first ultrasonic acoustic standing wave tofurther clarify the harvest fluid.
 15. The device of claim 1, whereinboth a planar acoustic standing wave and the multi-dimensional acousticstanding wave are created across the collection flow path.
 16. A methodfor separating biological cells from a fluid medium, comprising: flowingthe fluid medium containing the biological cells through an acousticperfusion device, the device comprising: an acoustic chamber; an inletport, an inlet flow path leading from the inlet port to the acousticchamber; an outlet port for recirculating the fluid medium and thebiological cells; at least one collection port for collecting harvestfluid; and at least one ultrasonic transducer below the at least onecollection port and at least one reflector opposite the at least oneultrasonic transducer, wherein the at least one ultrasonic transducerincludes a piezoelectric material driven by a voltage signal to create amulti-dimensional acoustic standing wave across a collection flow pathleading from the acoustic chamber to the at least one collection port;driving the at least one ultrasonic transducer to create the acousticstanding wave; and collecting a fluid enriched in cells from the outletport and collecting a harvest fluid depleted in cells from the at leastone collection port.
 17. The method of claim 16, wherein the outlet portis below the inlet port.
 18. The method of claim 16, wherein a flow ratethrough the collection flow path is at least one order of magnitudesmaller than a flow rate through the inlet flow path.
 19. The method ofclaim 16, wherein the inlet flow path leads from the inlet portdownwards towards the outlet port and then upwards to the acousticchamber, creating a recirculating fluid stream that is locallysubstantially tangential to the at least one acoustic standing wave. 20.The method of claim 19, wherein the recirculating fluid streamtransports away cells that are constantly dropping from an edge regionof the acoustic standing wave.
 21. The method of claim 16, wherein apressure rise and an acoustic radiation force on cells are generated atan edge of the acoustic standing wave to clarify fluid passing throughthe acoustic standing wave.
 22. The method of claim 16, wherein a flowrate of the fluid medium entering the device through the inlet port isabout 1 liter per minute and a flow rate of the harvest fluid depletedin cells exiting the device through the at least one collection port isabout 10 milliliters per minute.
 23. The method of claim 16, furthercomprising a secondary flow chamber in which the harvest fluid passesthrough a second acoustic standing wave having a frequency higher thanthe first ultrasonic acoustic standing wave to further clarify theharvest fluid.
 24. A flow device adapted to (i) receive a flowingmixture containing a primary fluid and cells; and (ii) to use a firstacoustic standing wave to continuously draw off a harvest fluid streamdepleted in cells from the flowing mixture, thereby changing the cellconcentration of the flowing mixture.
 25. The device of claim 24,wherein the first acoustic standing wave is a planar acoustic standingwave or a multi-dimensional acoustic standing wave, or wherein both aplanar wave and a multi-dimensional acoustic standing wave are createdacross a flow path to obtain the harvest fluid stream.
 26. The device ofclaim 24, wherein a pressure rise and an acoustic radiation force oncells are generated at an edge of the first acoustic standing wave toclarify the flowing mixture passing through the standing wave.
 27. Thedevice of claim 24, further comprising a secondary flow chamber in whichthe harvest fluid stream depleted in cells passes through a secondacoustic standing wave having a frequency higher than the first acousticstanding wave.
 28. A flow device, comprising: at least one inlet forreceiving a flowing mixture of a primary fluid and cells, an ultrasonictransducer that produces a first ultrasonic multi-dimensional acousticstanding wave and uses a pressure rise and an acoustic radiation forceon cells, generated at an edge of the first ultrasonic multi-dimensionalacoustic standing wave, to separate the flowing mixture into a primaryhigh cell concentration fluid stream and a secondary harvest fluidstream; an outlet port for the primary high cell concentration fluidstream; a bleed port for extracting a concentrated fluid/cell mixture;and at least one collection port for the secondary harvest fluid stream.29. The device of claim 28, further comprising a secondary flow chamberin which the secondary harvest fluid stream passes through a secondacoustic standing wave having a frequency higher than the firstultrasonic acoustic standing wave.
 30. The device of claim 29, where thesecondary flow chamber is utilized to further clarify the secondaryharvest fluid stream by trapping and agglomerating materials having asize of about 10 microns or less, such that the agglomerated materialseparates from the secondary harvest fluid stream due to gravity.